Stable plurilamellar vesicles, their preparation and use

ABSTRACT

A new and substantially improved type of lipid vesicle, called stable plurilamellar vesicles (SPLVs), are described, as well as the process for making the same. SPLVs are stable during storage and can be used in vivo for the sustained release of compounds and in the treatment of disease.

The present application is a continuation-in-part of applicant's priorcopending applications Ser. No. 463,900 by M. W. Fountain and R. P. Lenkfiled Feb. 4, 1983, and now abandoned; Ser. No. 447,247 by R. P. Lenk,M. W. Fountain, A. S. Janoff, filed Dec. 6, 1982, and now abandoned;Ser. No. 411,466 by M. W. Fountain, M. J. Ostro, M. Popescu, and R. P.Lenk, filed Aug. 25, 1982, and now abandoned; Ser. No. 362,995 by M. W.Fountain filed Mar. 29, 1982, and now abandoned; Ser. No. 362,994 by M.W. Fountain filed Mar. 29, 1982, and now abandoned which are hereinincorporated by reference.

TABLE OF CONTENTS

1. Field of the Invention

2. Background of the Invention

2.1. Liposomes

2.2. Uses of Liposomes

3. Summary of the Invention

4. Brief Description of the Figures

5. Detailed Description of the Invention

5.1. Preparation of SPLVs

5.2. Characterization of SPLVs

5.2.1. Stability of SPLVs in Storage

5.2.2. Stability of SPLVs in Other Environments

5.2.3. Characteristics of SPLVs Administered In Vivo

5.2.4. Electron Spin Resonance

5.2.5. Entrapment of Active Material by SPLVs

5.2.6. Interaction of SPLV with Cells

5.2.7. Buoyant Density of SPLVs

5.2.8. Volume of SPLVs

5.2.9. Osmotic Properties of SPLVs

5.3. Uses of SPLVs

5.3.1. Delivery of Bioactive Compounds

5.3.2. Treatment of Pathologies

6. Example: Preparation of SPLVs

6.1. SPLVs Containing Antibiotics

6.2. SPLVs Containing Other Membrane Constituents

6.3. SPLVs Containing Pilocarpine

6.4. SPLVs Prepared with and without BHT

7. Example: SPLV Mediated Delivery In Vitro

8. Example: Treatment of Intracellular Infections

8.1. Effect of a Single Treatment of B. Canis Infection UsingSPLV-Entrapped Antibiotic

8.2. Effect of Multiple Treatment of B. Canis Infection UsingSPLV-Entrapped Antibiotic

8.3. Effectiveness of Treatments Using MLVs as Compared to SPLVs

8.4. Effect of Various SPLV-entrapped Antibiotics on Treatment ofInfection

8.5. Treatment of Dogs Infected with B. Canis

8.6. Treatment of B. Abortus in Guinea Pigs

8.7. Treatment of B. Abortus Infection in Cows

9. Example: Treatment of Ocular Afflictions

9.1. Treatment of Infectious Keratoconjunctivitis in Mice

9.2. Treatment of Rabbit Conjunctiva Using SPLV-Entrapped Antibiotic

9.3. Treatment of Keratoconjunctivitis Resulting from SubcutaneousInfections

9.4. Evaluation of the Effectiveness of SPLVs as Compared to LiposomePreparations in the Treatment of Ocular Infections

10. Example: Treatment of Viral Infections

10.1 Treatment of Lethal Lymphocytic Choriomeningitis Virus Infectionsin Mice

1. FIELD OF THE INVENTION

This invention relates to liposomes and their uses as carriers indelivery systems. More specifically, it relates to a new type of lipidvesicle having unique properties which confer special advantages such asincreased stability and high entrapment efficiency.

The compositions and methods described herein have a wide range ofapplicability to fields such as carrier systems and targeted deliverysystems. The practice of the present invention is demonstrated herein byway of example for the treatment of brucellosis, the treatment of ocularinfections, and the treatment of lymphocytic meningitis virusinfections.

2. BACKGROUND OF THE INVENTION

2.1. Liposomes

Liposomes are completely closed bilayer membranes containing anentrapped aqueous phase. Liposomes may be any variety of unilamellarvesicles (possessing a single membrane bilayer) or multilamellarvesicles (onion-like structures characterized by concentric membranebilayers each separated from the next by a layer of water).

The original liposome preparations of Bangham et al. (1965, J. Mol.Biol. 13:238-252) involved suspending phospholipids in an organicsolvent which was then evaporated to dryness leaving a waxy deposit ofphospholipid on the reaction vessel. Then an appropriate amount ofaqueous phase was added, the mixture was allowed to "swell", and theresulting liposomes which consisted of multilamellar vesicles(hereinafter referred to as MLVs) were dispersed by mechanical means.The structure of the resulting membrane bilayer is such that thehydrophobic (non-polar) "tails" of the lipid orient toward the center ofthe bilayer while the hydrophilic (polar) "heads" orient towards theaqueous phase. This technique provided the basis for the development ofthe small sonicated unilamellar vesicles (hereinafter referred to asSUVs) described by Papahadjapoulos and Miller (1967, Biochim. Biophys.Acta. 135:624-638). These "classical liposomes", however, had a numberof drawbacks not the least of which was a low volume of entrappedaqueous space per mole of lipid and a restricted ability to encapsulatelarge macromolecules.

Efforts to increase the entrapped volume involved first forming inversemicelles or liposome precursors, i.e., vesicles containing an aqueousphase surrounded by a monolayer of lipid molecules oriented so that thepolar head groups are directed towards the aqueous phase. Liposomeprecursors are formed by adding the aqueous solution to be entrapped toa solution of polar lipid in an organic solvent and sonicating. Theliposome precursors are then evaporated in the presence of excess lipid.The resultant liposomes, consisting of an aqueous phase entrapped by alipid bilayer are dispersed in aqueous phase (see U.S. Pat. No.4,224,179 issued Sept. 23, 1980 to M. Schneider).

In another attempt to maximize the efficiency of entrapmentPapahadjopoulos (U.S. Pat. No. 4,235,871 issued Nov. 25, 1980) describesa "reverse-phase evaporation process" for making oligolamellar lipidvesicles also known as reverse-phase evaporation vesicles (hereinafterreferred to as REVs). According to this procedure, the aqueous materialto be entrapped is added to a mixture of polar lipid in an organicsolvent. Then a homogeneous water-in-oil type of emulsion is formed andthe organic solvent is evaporated until a gel is formed. The gel is thenconverted to a suspension by dispersing the gel-like mixture in anaqueous media. The REVs produced consist mostly of unilamellar vesiclesand some oligolamellar vesicles which are characterized by only a fewconcentric bilayers with a large internal aqueous space. Certainpermeability properties of REVs were reported to be similar to those ofMLVs and SUVs (see Szoka and Papahadjopoulos, 1978, Proc. Natl. Acad.Sci. U.S.A. 75:4194-4198).

Liposomes which entrap a variety of compounds can be prepared, however,stability of the liposomes during storage is invariably limited. Thisloss in stability results in leakage of the entrapped compound from theliposomes into the surrounding media, and can also result incontamination of the liposome contents by permeation of materials fromthe surrounding media into the liposome itself. As a result the storagelife of traditional liposomes is very limited. Attempts to improvestability involved incorporating into the liposome membrane certainsubstances (hereinafter called "stabilizers") which affect the physicalproperties of the lipid bilayers (e.g., steroid groups). However, manyof these substances are relatively expensive and the production of suchliposomes is not cost-effective.

In addition to the storage problems of traditional liposomes a number ofcompounds cannot be incorporated into these vesicles. MLVs can only beprepared under conditions above the phase-transition temperature of thelipid membrane. This precludes the incorporation of heat labilemolecules within liposomes that are composed of phospholipids whichexhibit desirable properties but possess long and highly saturated sidechains.

2.2. Uses of Liposomes

Application of liposomes to therapeutic uses is described in Liposomes:From Physical Structures To Therapeutic Applications, Knight, ed.Elsevier, North-Holland Biomedical Press, 1981. Much has been writtenregarding the possibilities of using these membrane vesicles for drugdelivery systems though a number of problems with such systems remain.See, for example, the disclosures in U.S. Pat. No. 3,993,754 issued onNov. 23, 1976, to Yneh-Erh Rahman and Elizabeth A. Cerny, and U.S. Pat.No. 4,145,410 issued on Mar. 20, 1979, to Barry D. Sears. In a liposomedrug delivery system the medicament is entrapped during liposomeformation and then administered to the patient to be treated. Themedicament may be soluble in water or in a non-polar solvent. Typical ofsuch disclosures are U.S. Pat. No. 4,235,871 issued Nov. 25, 1980, toPapahadjopoulos and Szoka and U.S. Pat. No. 4,224,179, issued Sept. 23,1980 to M. Schneider.

Some desirable features of drug delivery systems are resistance to rapidclearance of the drug accompanied by a sustained release of the drugwhich will prolong the drug's action. This increases effectiveness ofthe drug and allows the use of fewer administrations. Some of theproblems encountered in using liposome preparations in vivo include thefollowing: (1) Liposome entrapped materials leak when the liposomes areincubated in body fluids. This has been attributed to the removal of theliposomal phospholipids by plasma high density lipoproteins (HDL), or tothe degradation of the liposome membrane by phospholipases, among otherreasons. A result of the degradation of the liposomes in vivo is thatalmost all the liposomal contents are released in a short period oftime, therefore, sustained release and resistance of the drug toclearance are not achieved. (2) On the other hand, if a very stableliposome is used in vivo (i.e., liposomes which do not leak whenincubated in body fluids), then the liposomal contents will not bereleased as needed. As a result, these stable liposomes are ineffectiveas carriers of therapeutic substances in vivo because the sustainedrelease or the ability to release the liposomal contents when necessaryis not accomplished. However, if one is treating an intracellularinfection, the maintenance of stability in biological fluids until thepoint that the liposome is internalized by the infected cell, iscritical. (3) The cost-effectiveness of the liposome carriers used indelivery systems. For example, an improved method for the chemotherapyof leishmanial infections using liposome encapsulated anti-leishmanialdrug has been reported by Steck and Alving in U.S. Pat. No. 4,186,183issued on Jan. 29, 1980. The liposomes used in the chemotherapycontained a number of stabilizers which increased the stability of theliposomes in vivo. However, as previously mentioned, these stabilizersare expensive and the production of liposomes containing thesestabilizers is not cost-effective. (4) Ultimately, the problemencountered in the use of liposomes as carriers in drug delivery systemsis the inability to effect a cure of the disease being treated. Inaddition to the inability to resist rapid clearance and to effectsustained release, a number of other explanations for the inability tocure diseases are possible. For instance, if the liposomes areinternalized into target cells or phagocytic cells (e.g.,reticuloendothelial cells), they are cleared from the system rapidly,rendering the entrapped drug largely ineffective against diseases ofinvolving cells other than the RES. After phagocytosis, the liposomalcontents are packaged within lysosomes of the phagocytic cell. Veryoften the degradative enzymes contained within the lysosome will degradethe entrapped compound or render the compound inactive by altering itsstructure or cleaving the compound at its active site. Furthermore, theliposomes may not deliver a dose which is effective due to the lowefficiency of entrapment of active compound into the vesicles whenprepared.

Liposomes have also been used by researchers as model membrane systemsand have been employed as the "target cell" in complement mediatedimmunoassays. However, when used in such assays, it is important thatthe liposome membrane does not leak when incubated in sera because theseassays measure the release of the liposome contents as a function ofserum complement activation by immune complex formation involvingcertain immunoglobulin classes (e.g., IgM and certain IgG molecules).

3. SUMMARY OF THE INVENTION

This invention presents a new and substantially improved type of lipidvesicles which hereinafter will be referred to as stable plurilamellarvesicles (SPLVs). Aside from being structurally different thanmultilamellar vesicles (MLVs), SPLVs are also prepared differently thanMLVs, possess unique properties when compared to MLVs, and present avariety of different advantages when compared to such MLVs. As a resultof these differences, SPLVs overcome many of the problems presented byconventional lipid vesicles heretofore available.

A heterogeneous mixture of lipid vesicles is realized when SPLVs aresynthesized. Evidence indicates that the lipids in the SPLVs areorganized in a novel supramolecular structure. Many of the lipidvesicles possess a high number of bilayers, occasionally as high as onehundred layers. It may be possible that this high degree of layeringcontributes to many of the surprising properties possessed by SPLVs,although the explanations are theoretical.

The properties of SPLVs include: (1) the ability to cure certaindiseases which other methodologies cannot cure; (2) greatly increasedstability of the SPLVs during storage in buffer; (3) the increasedability of SPLVs to withstand harsh physiologic environments; (4) theentrapment of materials at a high efficiency; (5) the ability to stickto tissues and cells for prolonged periods of time; (6) the ability torelease of entrapped materials slowly in body fluids; (7) the deliveryand ultimate dispersal of the liposomal contents throughout the cytosolof the target cell; (8) improved cost-effectiveness in preparation; and(9) release of compounds in their bioactive forms in vivo.

Due to the unique properties of SPLVs they are particularly useful ascarriers in delivery systems in vivo because they are resistant toclearance and are capable of sustained release. Methods for the use ofSPLVs for the delivery of bioactive compounds in vivo and the treatmentof pathologies, such as infections, are described.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 graphically demonstrates the difference in membrane stability (asreflected by % leakage) between MLVs and SPLVs treated with varyingconcentrations of urea.

FIG. 2 graphically represents the retention of both the lipid andaqueous phases of SPLVs in eyelid tissues of mice, and the sustainedrelease of gentamycin from the SPLVs in vivo.

FIG. 3 represents the electron spin resonance absorption spectrum ofSPLVs (A) compared to that of MLVs (B).

FIG. 4 graphically demonstrates the difference in the ability ofascorbate to reduce doxyl spin probes in SPLVs and in MLVs.

FIG. 5 graphically represents the effectiveness of a two stage treatmentof Brucella canis infections in mice using SPLV-entrapped streptomycinbased on B. canis recoverable from spleens of infected mice.

FIG. 6 graphically represents the effectiveness of a two stage treatmentof B. canis infections in mice using SPLV-entrapped streptomycin basedon B. canis recoverable from organs of infected mice.

FIG. 7 graphically represents the effectiveness of a two stage treatmentof Brucella abortus in guinea pigs using SPLV-entrapped streptomycin.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1. Preparation of SPLVs

SPLVs are prepared by a process which results in a product unique fromany other liposome previously described.

SPLVs are lipid vesicles possessing from a few to over one hundred lipidbilayers. The membrane bilayer is composed of a bimolecular layer of anamphipathic lipid in which the non-polar hydrophobic hydrocarbon "tails"point inward towards the center of the bilayer and the polar,hydrophilic "heads" point towards the aqueous phase. Occluded by thebilayers is an aqueous compartment, part of wich makes up the lumen ofthe vesicle, and part of which lies between adjacent layers. Complexedwith the lipid bilayers can be a variety of proteins, glycoproteins,glycolipids, mucopolysaccharides, and any other hydrophobic and/oramphipathic substance.

SPLVs are prepared as follows: An amphipathic lipid or mixture of lipidsis dissolved in an organic solvent. Many organic solvents are suitable,but diethyl ether, fluorinated hydrocarbons and mixtures of fluorinatedhydrocarbons and ether are preferred. To this solution are added anaqueous phase and the active ingredient to be entrapped. This biphasicmixture is converted to SPLVs by emulsifying the aqueous material withinthe solvent while evaporating the solvent. Evaporation can beaccomplished during sonication by any evaporative technique, e.g.,evaporation by passing a stream of inert gas over the mixture, byheating, or by vacuum. The volume of solvent used must exceed theaqueous volume by a sufficient amount so that the aqueous material canbe completely emulsified in the mixture. In practice, a minimum ofroughly 3 volumes of solvent to 1 volume of aqueous phase may be used.In fact the ratio of solvent to aqueous phase can vary to up to 100 ormore volumes of solvent to 1 volume aqueous phase. The amount of lipidmust be sufficient so as to exceed that amount needed to coat theemulsion droplets (about 40 mg of lipid per ml of aqueous phase). Theupper boundary is limited only by the practicality ofcost-effectiveness, but SPLVs can be made with 15 gm of lipid per ml ofaqueous phase.

The process produces lipid vesicles with different supermolecularorganization than conventional liposomes. According to the presentinvention, the entire process can be performed at a temperature range of4°-60° C. regardless of the phase transition temperature of the lipidused. The advantage of this latter point is that heat labile productswhich have desirable properties, for example, easily denatured proteins,can be incorporated in SPLVs prepared from phospholipid such asdistearoylphosphatidylcholine, but can be formed into conventionalliposomes only at temperatures above their phase-transition-temperature.The process usually allows more than 20% of the available water solublematerial to be encapsulated and more than 40% of the available lipidsoluble material to be encapsulated. With MLVs the entrapment of aqueousphase usually does not exceed 10%.

Most amphipathic lipids may be constituents of SPLVs. Suitablehydrophilic groups include but are not limited to: phosphato,carboxylic, sulphato and amino groups. Suitable hydrophobic groupsinclude but are not limited to: saturated and unsaturated aliphatichydrocarbon groups and aliphatic hydrocarbon groups substituted by atleast one aromatic and/or cycloaliphatic group. The preferredamphipathic compounds are phospholipids and closely related chemicalstructures. Examples of these include but are not limited to: lecithin,phosphatidylethonolamine, lysolecithin, lysophatidylethanolamine,phosphatidylserine, phosphatidylinositol, sphingomyelin, cardiolipin,phosphatidic acid and the cerebrosides. Specific examples of suitablelipids useful in the production of SPLVs are phospholipids which includethe natural lecithins (e.g., egg lecithin or soybean lecithin) andsynthetic lecithins, such as saturated synthetic lecithins (e.g.,dimyristoylphosphatidylcholine, or dipalmitoyl-phosphatidylcholine ordistearoylphosphatidylcholine) and unsaturated synthetic lecithins(e.g., dioloyl-phosphatidylcholine or dilinoloylphosphatidylcholine. TheSPLV bilayers can contain a steroid component such as cholesterol,coprostanol, cholestanol, cholestane and the like. When using compoundswith acidic hydrophilic groups (phosphato, sulfato, etc.) the obtainedSPLVs will be anionic; with basic groups such as amino, cationicliposomes will be obtained; and with polyethylenoxy or glycol groupsneutral liposomes will be obtained. The size of the SPLVs varies widely.The range extends from about 100 nm to about 10,000 nm (10 microns) andusually about 100 nm to about 1,500 nm.

Virtually any bioactive compound can be entrapped within a SPLV(entrapped is defined as entrapment within the aqueous compartment orwithin the membrane bilayer). Such compounds include but are not limitedto nucleic acids, polynucleotides, antibacterial compounds, antiviralcompounds, antifungal compounds, anti-parasitic compounds, tumoricidalcompounds, proteins, toxins, enzymes, hormones, neurotransmitters,glycoproteins, immunoglobulins, immunomodulators, dyes, radiolabels,radio-opaque compounds, fluorescent compounds, polysaccharides, cellreceptor binding molecules, anti-inflammatories, antiglaucomic agents,mydriatic compounds, local anesthetics, etc.

The following is an example of the proportions that may be used in SPLVsynthesis: SPLVs may be formed by adding 50 micromoles of phospholipidto 5 ml of diethyl ether containing 5 micrograms of BHT(butylatedhydroxytoluene) and then adding 0.3 ml of aqueous phasecontaining the active substance to be encapsulated. The resultantsolution which comprises the material to be entrapped and the entrappinglipid is sonicated while streaming an inert gas over the mixture thusremoving most of the solvent. This embodiment produces particularlystable SPLVs partially because of the incorporation of BHT into thevesicles.

See also Lenk, et al., 1982, Eur. J. Biochem. 121:475-482 whichdescribes a process for making liposome-encapsulated antibodies bysonicating and evaporating a solution of cholesterol andphosphatidylcholine in a mixture of chloroform and ether with aqueousphase added, but does not set forth the relative proportions of lipid toaqueous phase.

5.2. Characterization of SPLVs

SPLVs are clearly distinct in their properties from liposomes with asingle or several lamellae (e.g., SUVs, and REVs). Freeze-fractureelectron microscopy indicates that SPLV preparations are substantiallyfree of SUVs and REVs, that is, less than 20% of the vesicles areunilamellar. They are, however, indistinguishable from MLVs by electronmicroscopic techniques although many of their physical properties aredifferent. Thus, the following detailed comparison is focused ondistinguishing SPLVs from MLVs.

5.2.1. Stability of SPLVs in Storage

Stability of a lipid vesicle refers to the ability of the vesicle tosequester its occluded space from the external environment over a longperiod of time. For a lipid vesicle to be useful it is paramount that itbe stable in storage and handling. For some applications, however, it isdesirable that the vesicle leak its contents slowly when applied. Forother applications it is desirable that the vesicle remain intact afteradministration until it reaches its desired site of action. It will beseen that SPLVs demonstrate these desirable characteristics, while MLVsdo not.

There are two factors that cause vesicles to leak. One is auto-oxidationof the lipids whereby the hydrocarbon chains form peroxides whichdestabilize the bilayers. This oxidation can be drastically slowed downby the addition of antioxidants such as butylated hydroxy toluene (BHT)to the vesicle preparation. Vesicles can also leak because agents in theexterior environment disrupt the bilayer organization of the lipids suchthat the lipids remain intact, but the membrane develops a pore.

Preparations of lipid vesicles are white in color when first made. Uponauto-oxidation, the preparation becomes discolored (brownish). Acomparison of MLVs to SPLVs prepared using the same lipid and aqueouscomponents reveals that MLVs discolor within one to two weeks whereasSPLVs remain white for at least two months. This is supported by thinlayer chromatography of the constituent lipids which showed degradationof the lipids in the MLVs but not of the lipids of the SPLVs. When thesevesicles are prepared by adding BHT as well as the other constituents,then MLVs appear slightly discolored within one month whereas the SPLVsremain white and appear stable for at least 6 months and longer.

When placed in a buffer containing isotonic saline at neutral pH, SPLVscontaining antibiotic are stable for more than four months, asdemonstrated in Table I. These data indicate that none of the antibioticoriginally encapsulated within the SPLVs leaked out in the period of theexperiment.

Other evidence indicates that SPLVs are able to sequester anencapsulated agent from molecules as small as calcium ions for more thansix months. Arsenazo III is a dye which changes color from red to bluewith the slightest amount of divalent cation present. By encapsulatingthe dye in SPLVs and adding calcium chloride to the storage buffer it ispossible to measure the stability of the vesicles by looking for a colorchange. The color remains undetectably different from its original colorfor at least 6.5 months, demonstrating that neither has the dye leakedout nor the ion leaked in.

                  TABLE I                                                         ______________________________________                                        STABILITY OF EGG PHOSPHATIDYLCHOLINE                                          SPLVs AFTER STORAGE IN SEALED                                                 CONTAINERS AT 4° C. FOR 41/2 MONTHS.sup.a                                         Initial    Leakage    Bioavailability                              Entrapped  Entrapment Into       of Entrapped                                 Drug       %          Supernatant.sup.b                                                                        Drug (%)                                     ______________________________________                                        Streptomycin                                                                             34.1       0          97                                           Sulfate                                                                       Spectinomycin                                                                            37.2       0          84                                           Chloramphenicol                                                                          35.2       0          89                                           Oxytetracycline                                                                          18.8       0          91                                           Erythromycin                                                                              0.4       0          97                                           Sulfamerazine                                                                             6.3       0          93                                           ______________________________________                                         .sup.a SPLVs were prepared using 127 μM egg phosphatidylcholine (EPC)      and 25 μM drug. At the end of 41/2 months storage at 4° C. the      SPLVs were separated from storage buffer by centrifugation. Serial            dilutions of the SPLV contents and the supernatant were applied to            bacterial lawns in order to determine bioactivity as compared to standard     dilutions of antibiotic.                                                      .sup.b 0 indicates below detectable levels                               

These experiments demonstrate that SPLVs are sufficiently stable towithstand storage and handling problems. Although it is possible to makeMLVs which are stable for this long, they must be made from syntheticlipids such as DSPC and thus become prohibitively expensive.

5.2.2. Stability of SPLVs in Other Environments

Placing lipid vesicles in a medium which contains membrane perturbingagents is a way to probe different molecular organizations. Depending onhow the membrane is organized, different vesicles will responddifferently to such agents.

In the following experiments vesicles were prepared which contained aradioactive tracer molecule (³ H inulin) within the occluded aqueouscompartment. Inulin, a polysaccharide, partitions into the aqueousphase, and thus when radiolabeled may be used to trace the aqueouscontents of lipid vesicles. After an appropriate interval of exposure toa given agent, the vesicles were separated from the medium bycentrifugation, and the relative amount of radioactivity that escapedfrom the vesicles into the medium was determined. These results arereported in Table II; values are expressed as percent leaked, meaningthe proportion of radioactive material in the surrounding mediumrelative to the starting amount encapsulated in the vesicles.

SPLVs are more stable than MLVs in hydrochloric acid. Table IIillustrates that both MLVs and SPLVs, when made from egg lecithin, aredestabilized when exposed to 0.125 N hydrochloric acid for one hour.However, it is noteworthy that the SPLVs are considerably lesssusceptible to the acid than MLVs. Presumably this different responsereflects an intrinsic difference in the way the lipids interact withtheir environment.

                  TABLE II                                                        ______________________________________                                        STABILITY OF SPLVS IN OTHER ENVIRONMENTS                                                      % LEAKAGE                                                     Incubating Medium.sup.a                                                                         MLVs    SPLVs                                               ______________________________________                                        Hydrochloric Acid                                                             0.125 M           90.5    55.2                                                Urea                                                                          1 M               21.7    44.8                                                Guanidine                                                                     0.5 M              5.7     7.4                                                1.0 M              8.3    10.1                                                Ammonium Acetate                                                              0.5 M             27.0    67.0                                                1.0 M             25.9    54.7-63.1                                           Serum             76.2    57.8                                                ______________________________________                                         .sup.a Incubation time is 2 to 4 hours except incubation in HCl was for 1     hour.                                                                    

SPLVs also respond differently than MLVs when exposed to urea (FIG. 1and Table II). Urea is a molecule with both a chaotropic effect(disrupts the structure of water) and a strong dipole moment. It isobserved that SPLVs are far more susceptible to urea than they are to anosmotic agent such as sodium chloride at the same concentration (FIG.1). MLVs do not leak significantly more in urea than they would insodium chloride. Although the explanations for this different behaviorare theoretical, it would appear that the response is due to the dipoleeffect, rather than a chaotropic property, since guanidine, a moleculesimiliar to urea, does not destabilize SPLVs (Table II). Althoughguanidine is also strongly chaotropic, it does not possess a strongdipole moment.

SPLVs are also susceptible to ammonium acetate, while MLVs are not(Table II). However, neither ammonium ion (in ammonium chloride) noracetate (in sodium acetate) are particularly effective in causing SPLVsto destabilize. Thus it would appear that it is not the ion itself, butthe polarity of the ammonium acetate which is responsible for inducingleakage.

Initially these results seem surprising because SPLVs are much morestable than MLVs when incubated in body fluids such as sera or blood.However a theoretical explanation for these results can be proposed (ofcourse other explanations are possible). If the stability of the SPLV isdue to the unique structure of its membrane bilayers such that the polargroups of the membrane lipids are hydrated by a cloud of oriented watermolecules, or hydration shell, then it is possible that any agent whichdisrupts or interferes with such hydration shells would promote changesin structural membrane integrity, and therefore, leakage.

Independent of the theoretical explanations for the destabilization ofSPLVs in urea, the results serve to demonstrate characteristicdifferences between the structure of MLVs and SPLVs. This differenceserves a very useful purpose in application. As described infra, SPLVsbecome slowly leaky when applied to the eye. Presumably this desiredslow release of contents is due to a similar destabilization of theSPLVs when exposed to tear fluid.

SPLVs are more stable in serum than MLVs. Many applications of lipidvesicles include administering them intraperitoneally, such as for thetreatment of brucellosis. To be effective, the vesicles must survive fora sufficient time to reach their desired target. SPLVs and MLVs, bothmade from egg lecithin, were exposed to fetal bovine serum whichcontained active complement, (Table II). After 48 hours exposure at 37°C., SPLVs are demonstrably more stable than MLVs.

5.2.3. Characteristics of SPLVs Administered in Vivo

SPLVs demonstrate a number of characteristics which make themparticularly suitable as carriers for delivery systems in vivo:

(A) SPLVs are resistant to clearance. When SPLVs are administered to anorganism both the lipid component and the entrapped active ingredientare retained in the tissues and by the cells to which they areadministered;

(B) SPLVs can be engineered to provide sustained release. The stabilityof SPLVs is "adjustable" in that SPLVs are very stable during storageand are stable in the presence of body fluids but when administered invivo a slow leakage of the active ingredient permits the sustainedrelease of the active ingredient;

(C) Because of the high level of entrapment and stability whenadministered, effective doses of the active ingredient are released; and

(D) The production of SPLVs is very cost effective in that stability ofthe vesicles is achieved without incorporating expensive stabilizersinto the bilayers.

The following experiments demonstrate some of these characteristics ofSPLVs when administered topically onto the eyes of test animals. TheSPLVs used in these experiments were prepared as previously describedexcept that the lipid bilayer and the active ingredient were eachradiolabeled in order to trace these components in the eye tissues overa period of time.

SPLVs were prepared using 100 mg egg phosphatidylcholine (EPC) and 100mg gentamycin sulfate. The lipid component was radiolabeled by theincorporation of trace amounts of ¹²⁵ I-phosphatidylethanolamine (¹²⁵I-PE) into the bilayers, whereas the active ingredient in the aqueousphase was radiolabeled by the addition of ¹²⁵ I-gentamycin sulfate (¹²⁵I-GS). The SPLVs were washed with buffer repeatedly in order toeffectively remove unincorporated or unencapsulated materials.

An aliquot of the SPLV preparation was removed and extracted in order toseparate the organic phase from the aqueous phase. The radioactivity ofeach phase was measured in order to determine the initial ratio of ¹²⁵I-PE:¹²⁵ I-GS (cpm (counts per minute) in the lipid phase:cpm in theaqueous phase) which was incorporated into the SPLVs.

The extraction was done as follows: 0.8 ml of 0.4 M NaCl (aqueous), 1 mlchloroform, and 2 ml methanol were mixed to form a homogeneous phase.Then 4 μl of the radiolabeled SPLVs were added and mixed; as the SPLVcomponents dissolved into the organic phase and into the aqueous phase,the mixture, which was initially turbid, became clear. The phases wereseparated by adding and mixing 1 ml 0.4 M NaCl (aqueous) and 1 mlchloroform, which was then centrifuged at 2,800×g for 5 minutes. Analiquot (1 ml) of each phase was removed and the radioactivity (in cpm)was measured. (The initial ratio of ¹²⁵ I-PE:¹²⁵ I-GS was 1.55:1).

Fifteen adult female Swiss Webster mice were anesthetized and restrained(in order to prevent them from wiping their eyes). Equal aliquots (2 μl)of the radiolabeled SPLVs in suspension were topically applied to eacheye. Groups of three animals were then sacrificed at each of thefollowing points: 1, 2, 3, 18, and 24 hours. Nine female Swiss Webstermice (controls) were treated identically except that equal aliquots (2μl) of an aqueous solution of radiolabeled gentamycin sulfate wereapplied topically to each eye. Groups of three control animals weresacrificed at the end of 1, 4, and 8 hours.

Immediately after sacrifice the eyelids of the animals were removed,minced, and extracted (using the procedure previously described) inorder to separate the aqueous components from the lipid components. Theradioactivity of such phase was determined (as well as the total numberof radioactive counts recovered). The radioactivity measured in thelipid phase is an indication of the retention of SPLV lipids by the eyetissue, whereas the radioactivity measured in the aqueous phase is anindication of the retention of gentamycin in the eye tissue. FIG. 2graphically demonstrates the retention of each component in the eyelidtissue (expressed as the percent of the original number of cpm appliedto the eye).

FIG. 2 clearly demonstrates the retention of the SPLV lipid component inthe eyelid tissue over a 24 hour period, and the sustained release ofgentamycin from the SPLVs over a 24 hour period (as reflected by thepercent gentamycin retained in the eyelid tissue during this time). FIG.2 also demonstrates that unencapsulated gentamycin (aqueous gentamycinadministered topically) is rapidly cleared from the eyelid tissue. Forexample, gentamycin in solution (control) was cleared from the eyelidtissue within 4 hour (less than 5% of the gentamycin remained in theeyelid tissue). On the other hand, more than 50% of theSPLV-encapsulated gentamycin was retained by the eyelid tissue in this 4hour period; in fact, at the end of 24 hours more than 15% of theSPLV-encapsulated gentamycin was retained by the eyelid tissue. Thisindicates that approximately 85% of the SPLV-encapsulated gentamycin wasreleased over a 24 hour period whereas 95% of the unencapsulatedgentamycin sulfate was cleared within a 4 hour period.

Table III compares the ratio of the SPLV lipid phase:aqueous phaseretained in the eyelid tissue at each time point. An increase in thisratio indicates release of gentamycin from the SPLVs.

The bioactivity of the SPLV-encapsulated gentamycin sulfate which wasretained by the eyelid tissues was also evaluated. Gentamycin sulfatewas recovered from the eyelid tissues by removing an aliquot from theaqueous phase of the eyelid extracts prepared 3 hours after theSPLV-encapsulated gentamycin sulfate was applied to the eye. The aqueousphase was serially diluted and 2 μl aliquots were placed ontoStaphylococcus aureus lawns on agar plates; after 24 hours incubationthe zones of inhibition were measured. The gentamycin sulfate recoveredfrom the eyelid tissue extracts of animals treated withSPLV-encapsulated gentamycin sulfate fully retained its bioactivity.

                  TABLE III                                                       ______________________________________                                        SUSTAINED RELEASE OF SPLV-ENCAPSULATED                                        GENTAMYCIN AFTER TOPICAL                                                      APPLICATION IN EYES OF MICE                                                              Total SPLV Compo-                                                                            Ratio of SPLV Lipid:                                           nents Recovered                                                                              Aqueous Phase                                       Time       from Eyelids   Retained In Eyelids                                 Post-Application                                                                         (% Initial Dose)                                                                             (.sup.125 I-PE:.sup.125 I-GS)                       ______________________________________                                        0          100%           1.55                                                 1 hr      100%           2.1                                                  3 hr      100%           2.82                                                18 hr       94%           6.89                                                24 hr      85.1%          7.17                                                ______________________________________                                    

5.2.4. Electron Spin Resonance

Although SPLVs and MLVs appear identical by electron microscopy, ESR(electron spin resonance) spectroscopy reveals differences in theirsupramolecular structure. SPLVs can be distinguished from MLVs on thebasis of their molecular architecture as evidence by their increasedmolecular order, increased molecular motion and greater penetrability toascorbate. It is likely that these differences in molecular architecturecontribute to their different biological effects.

In electron spin resonance spectroscopy a spin probe such as 5-doxylstearate (5 DS) is incorporated into the lipid bilayer. The unpairedelectron of the doxyl group absorbs microwave energy when the sample isinserted into a magnetic field. The spectrum of the absorption allowsthe determination of three empirical parameters: S, the order parameter;A.sub.°, the hyperfine coupling constant; and Tau the rotationalcorrelation time. A typical reading is shown in FIG. 3, wherein A is theSPLV signal and B is the MLV signal, both are labeled with 5-doxylstearate. The spectra were taken at room temperature, scan range was 100Gauss. The order parameter(s) which is dependent on both 2T₁ and 2T₁₁measures the deviation of the observed ESR signal from the case of acompletely uniform orientation of the probe. For a uniformly orientedsample S=1.00, a random sample S=0. The hyperfine coupling constant,A.sub.°, which can be calculated from 2T₁ and 2T₁₁ is considered toreflect local polarity and thus reflects the position of the spin probewithin the membrane. The rotational correlation time (which is dependenton W_(o), h_(o), h-1) can be thought of as the time required for themolecules to "forget" what their previous spatial orientations were. Atypical ESR determination of the differences between SPLVs and MLVshaving 5-DS as the spin probe is summarized in Table IV.

Although in both cases the spin probe is reporting from the same depthin the bilayer, SPLVs possess a significantly greater degree ofmolecular order and molecular motion than MLVs.

Another illustration of the differences between SPLVs and MLVs residesin the ability of ascorbate to reduce doxyl spin probes. It has beenknown for some time that ascorbate reduces doxyl moieties presumably totheir hydroxylamine analogs which do not absorb microwave energy in amagnetic field. In aqueous solutions the reduction occurs rapidly withconcomitant loss of ESR signal. If the spin probe is in a protectedenvironment such as a lipid bilayer it may be reduced more slowly or notat all by the hydrophilic ascorbate.

                  TABLE IV                                                        ______________________________________                                        ESR CHARACTERIZATION OF SPLVS AND MLVS                                               Tau            S      A°                                        ______________________________________                                        SPLVs    2.65 × 10.sup.-9 Sec                                                                     0.614  14.9                                         MLVs     3.65 × 10.sup.-9 Sec                                                                     0.595  14.9                                         ______________________________________                                    

Thus the rate of nitroxide reduction can be used to study the rate ofpenetration of the ascorbate into lipid bilayers. FIG. 3 shows thepercentage remaining spin versus time for SPLVs and MLVs suspended in anascorbate solution. At 90 minutes the ascorbate has reduced 25% of theprobe embedded in MLVs but 60% of the probe embedded in SPLVs. SPLVsallow for a dramatically greater penetrability of ascorbate than doMLVs.

5.2.5. Entrapment of Active Material by SPLVs

As another prime example of the superiority of SPLVs over traditionalMLVs, SPLVs entrap a larger percentage of the available active materialthereby conserving material (see Table V).

5.2.6. Interaction of SPLVs with Cells

Still another benefit of SPLVs is that SPLVs interact with cells suchthat a relatively large portion of the materials encapsulated inside thevesicle is dispersed throughout the cytoplasm of the cells rather thanbeing limited to phagocytic vesicles.

                  TABLE V                                                         ______________________________________                                        COMPARISON OF MLVS AND SPLVS                                                                 % Available Material Entrapped                                 Encapsulation of:                                                                              MLVs        SPLVs                                            ______________________________________                                        inulin (aqueous  2-6%        20-30%                                           space marker)                                                                 bovine serum     15%         20-50%                                           albumin                                                                       streptomycin     12-15%      20-40%                                           polyvinylpyrrolidone                                                                            5%         25-35%                                           (aqueous space)                                                               ______________________________________                                    

When SPLVs are mixed with cells the two appear to coalesce. Bycoalescence, SPLVs, unlike MLVs, interact with cells in vitro so thatall the cells contain at least some of the materials originallyentrapped in the SPLVs. This material appears to be distributedthroughout each cell and not limited to just the phagocytic vesicles.This can be demonstrated by incorporating ferritin in the aqueous phaseof a SPLV preparation. After coalescence with a cell in culture,ultrastructural analysis reveals that the ferritin is distributedthroughout the cytosol and is not bound by intracellular membranes.While this phenomenon can be shown to occur with MLVs a greater quantityof material can be transferred by SPLVs.

5.2.7. Buoyant Density of SPLVs

Additionally, SPLVs have a lower buoyant density than MLVs. This ismeasured by banding in a ficol gradient (see Table VI).

5.2.8. Volume of SPLVs

Furthermore, when collected in a pellet by centrifugation from 1,000 to100,000×g, SPLVs form a pellet that is substantially larger than MLVs,given the same phospholipid concentration. At a force of 16,000×g, theSPLVs form a pellet approximately one third larger than MLVs.

5.2.9. Osmotic Properties of SPLVs

Since ospholipid bilayers are permeable to water, placing MLVs in ahypertonic environment drives the occluded water out due to osmoticforce. SPLVs shrink more than MLVs. In addition, after shrinking 16hours in a buffer that is twenty times higher than the internal saltconcentration, SPLVs do not shrink to the same final volume as MLVs(SPLV pellets remain 1/3 larger than MLV pellets). This indicates thatthe difference in pellet size is not due to differences in aqueousenclosed volume.

5.3. Uses of SPLVs

SPLVs are particularly useful in systems where the following factors areimportant: stability during storage and contact with body fluids; arelatively high degree of encapsulation; cost-effectiveness; and therelease of the entrapped compound in its biologically active form.

                  TABLE VI                                                        ______________________________________                                        BUOYANT DENSITY                                                                         MLVs       SPLVs                                                    ______________________________________                                        in ficol    layers above 1%                                                                            layers above 0.5%                                    in gms/ml   1.071        1.0274                                               ______________________________________                                    

Furthermore, depending upon the mode of administration in vivo, SPLVscan be resistant to rapid clearance (e.g., where sustained delivery isimportant) or can be delivered to the cells of the RES.

As a result, the SPLVs of the invention are usefully employed in a widevariety of systems. They may be used to enhance the therapeutic efficacyof medications, to cure infections, to enhance enzyme replacement, oraldrug delivery, topical drug delivery, for introducing geneticinformation into cells in vitro and in vivo, for the production ofvaccines, for the introduction of recombinant deoxyribonucleic acidsegments into cells, or as diagnostic reagents for clinical testsfollowing release of entrapped "reporter" molecules. The SPLVs can alsobe employed to encapsulate cosmetic preparations, pesticides, compoundsfor sustained slow release to effect the growth of plants and the like.

The methods which follow, while described in terms of the use of SPLVs,contemplate the use of SPLVs or any other liposome or lipid vesiclehaving functional characteristics similar to those of SPLVs.

5.3.1. Delivery of Bioactive Compounds

Delivery of compounds to cells in vitro (e.g., animal cells, plantcells, protists, etc.) generally requires the addition of the SPLVscontaining the compound to the cells in culture. SPLVs, however, canalso be used to deliver compounds in vivo in animals (including man),plants and protists. Depending upon the purpose of delivery, the SPLVsmay be administered by a number of routes: in man and animals thisincludes but is not limited to injection (e.g., intravenous,intraperitoneal, intramuscular, subcutaneous, intraauricular,intramammary, intraurethrally, etc.), topical application (e.g., onafflicted areas), and by absorption through epithelial or mucocutaneouslinings (e.g., ocular epithelia, oral mucosa, rectal and vaginalepithelial linings, the respiratory tract linings, nasopharyngealmucosa, intestinal mucosa, etc.); in plants and protists this includesbut is not limited to direct application to organism, dispersion in theorganism's habitat, addition to the surrounding environment orsurrounding water, etc.

The mode of application may also determine the sites and cells in theorganism to which the compound will be delivered. For instance, deliveryto a specific site of infection may be most easily accomplished bytopical application (if the infection is external). Delivery to thecirculatory system (and hence reticuloendothelial cells), may be mosteasily accomplished by intravenous, intraperitoneal, intramuscular, orsubcutaneous injections.

Since SPLVs allow for a sustained release of the compound, doses whichmay otherwise be toxic to the organism may be utilized in one or moreadministrations to the organism.

The sections which follow describe some overall schemes in which SPLVsmay be used and demonstrate but do not limit the scope of the presentinvention.

5.3.2. Treatment of Pathologies

A number of pathological conditions which occur in man, animals andplants may be treated more effectively by encapsulating the appropriatecompound or compounds in SPLVs. These pathologic conditions include butare not limited to infections (intracellular and extracellular), cysts,tumors and tumor cells, allergies, etc.

Many strategies are possible for using SPLVs in the treatment of suchpathologies; a few overall schemes are outlined below which areparticularly useful in that they take advantage of the fact that SPLVswhen administrered in vivo are internalized by macrophages.

In one scheme, SPLVs are used to deliver therapeutic agents to sites ofintracellular infections. Certain diseases involve an infection of cellsof the reticuloendothelical system, e.g., brucellosis. Theseintracellular infections are difficult to cure for a number of reasons:(1) because the infectious organisms reside within the cells of thereticuloendothelial system, they are sequestered from circulatingtherapeutic agents which cannot cross the cell membrane intherapeutically sufficient concentrations, and, therefore, are highlyresistant to treatment; (2) often the administration of toxic levels oftherapeutic agents are required in order to combat such infections; and(3) the treatment has to be completely effective because any residualinfection after treatment can reinfect the host organism or can betransmitted to other hosts.

According to one mode of the present invention, SPLVs containing anappropriate biologically active compound are administered (preferablyintraperitoneally or intravenously) to the host organism or potentialhost organism (e.g., in animal herds, the uninfected animals as well asinfected animals may be treated). Since phagocytic cells internalizeSPLVs, the administration of an SPLV-encapsulated substance that isbiologically active against the infecting organism will result indirecting the bioactive substance to the site of infection. Thus, themethod of the present invention may be used to eliminate infectioncaused by a variety of microorganisms, bacteria, parasites, fungi,mycoplasmas, and viruses, including but not limited to: Brucella spp.,Mycobacterium spp., Salmonella spp., Listeria spp., Francisella spp.,Histoplasma spp., Corynebacterium spp., Coccidiodes spp. and lymphocyticchoriomeningitis virus.

The therapeutic agent selected will depend upon the organism causing theinfection. For instance, bacterial infections may be eliminated byencapsulating an antibiotic. The antibiotic can be contained within theaqueous fluid of the SPLV and/or inserted into the vesicle bilayer.Suitable antibiotics include but are not limited to: penicillin,ampicillin, hetacillin, carbencillin, tetracycline, tetracyclinehydrochloride, oxytetracycline hydrochloride, chlortetracyclinehydrochloride, 7-chloro-6-dimethyltetracycline, doxycycline monohydrate,methacycline hydrochloride, minocycline hydrochloride, rolitetracycline,dihydrostreptomycin, streptomycin, gentamicin, kanamycin, neomycin,erythromycin, carbomycin, oleandomycin, troleandomycin, Polymyxin Bcollistin, cephalothin sodium, cephaloridine, cephaloglycin dihydrate,and cephalexin monohydrate.

We have demonstrated the effectiveness of such treatments in curingbrucellosis (see Examples, infra). By the procedure of this invention,the effectiveness and duration of action are prolonged. It is surprisingthat this system is effective for treating infections which do notrespond to known treatments such as antibiotics entrapped in MLVs.Successful treatment is unexpected since any small remaining infectionswill spread and the infectious cycle will commence again. We have alsodemonstrated success in treating lymphocytic choriomeningitis virusinfection.

Of course, the invention is not limited to treatment of intracellularinfections. The SPLVs can be directed to a variety of sites of infectionwhether intracellular or extracellular. For instance, in anotherembodiment of the present invention, macrophages are used to carry anactive agent to the site of a systemic extracellular infection.According to this scheme, SPLVs are used to deliver a therapeuticsubstance to uninfected macrophages by administering the SPLVs in vivo(preferably intraperitoneally or intravenously). The macrophages willcoalesce with the SPLVs and then become "loaded" with the therapeuticsubstance; in general, the macrophages will retain the substance forapproximtely 3 to 5 days. Once the "loaded" macrophages reach the siteof infection, the pathogen will be internalized by the macrophages. As aresult, the pathogen will contact the therapeutic substance containedwithin the macrophage, and be destroyed. This embodiment of theinvention is particularly useful in the treatment of Staphylococcusaureus mastitis in man and cattle.

If the site of infection or affliction is external or accessible theSPLV-entrapped therapeutic agent can be applied topically. Aparticularly useful application involves the treatment of eyeafflictions. In the case of ocular afflictions, SPLVs containing one ormore appropriate active ingredients may be applied topically to theafflicted eye. A number of organisms cause eye infections in animals andman. Such organisms include but are not limited to: Moraxella spp.,Clostridium spp., Corynebacterium spp., Diplococcus spp., Flavobacteriumspp., Hemophilus spp., Klebsiella spp., Leptospira spp., Mycobacteriumspp., Neisseria spp., Propionibacterium spp., Proteus spp., Pesudomonasspp., Serratia spp., Escherichia spp., Staphylococcus spp.,Streptococcus spp. and bacteria-like organisms including Mycoplasma spp.and Rickettsia spp. These infections are difficult to eliminate usingconventional methods because any residual infection remaining aftertreatment can reinfect through lacrimal secretions. We have demonstratedthe use of SPLVs in curing ocular infections caused by Moraxella bovis(see examples, infra).

Because SPLVs are resistant to clearance and are capable of sustainedrelease of their contents, SPLVs are also useful in the treatment of anyaffliction requiring prolonged contact with the active treatingsubstance. For example, glaucoma is a disorder characterized by agradual rise in intraocular pressure causing progressive loss ofperipheral vision, and, when uncontrolled, loss of central vision andultimate blindness. Drugs used in the treatment of glaucoma may beapplied topically as eyedrops. However, in some cases treatment requiresadministering drops every 15 minutes due to the rapid clearing of thedrug from the eye socket. If an affliction such as glaucoma is to betreated by this invention therapeutic substances as pilocarpine,Floropryl, physostigmine, carcholin, acetazolamide, ethozolamide,dichlorphenamide, carbachol, demecarium bromide,diisopropylphosphofluoridate, ecothioplate iodide, physostigmine, orneostigmine, etc. can be entrapped within SPLVs which are then appliedto the affected eye.

Other agents which may be encapsulated in SPLVs and applied topicallyinclude but are not limited to: mydriatics (e.g., epinephrine,phenylepinephrine, hydroxy amphetamine, ephedrine, atropine,homatropine, scopolamine, cyclopentolate, tropicamide, encatropine,etc.); local anesthetics; antiviral agents (e.g., idoxuridine, adeninearabinoside, etc.); antimycotic agents (e.g., amphoteracin B, natamycin,pimaricin, flucytosine, nystantin, thimerosal, sulfamerazine,thiobendazole, tolnaftate, grisiofulvin, etc.); antiparasitic agents(e.g., sulfonamides, pyrimethamine, clindamycin, etc.); andanti-inflammatory agents (e.g., corticosteriods such as ACTH,hydrocortisone, prednisone, medrysone, beta methasone, dexamethasone,fluoromethalone, triamcinalone, etc.).

The following Examples are given for purposes of illustration and not byway of limitation on the scope of the invention.

6. EXAMPLE: PREPARATION OF SPLVS

As explained in Section 5.1. the basic method for preparing SPLVsinvolves dissolving a lipid or mixture of lipids into an organicsolvent, adding an aqueous phase and the material to be encapsulated,and sonicating the mixture. In the preferred embodiment the solvent isremoved during sonication; however, the organic solvent may be removedduring or after sonication by any evaporative technique. The SPLVs usedin all of the examples contained herein were prepared as described inthe following subsections (however any method which yields SPLVs may beused).

6.1. SPLVs Containing Antibiotics

A 5 ml diethyl ether solution of 100 mg lecithin was prepared. Themixture was placed in a round-bottom flask. Then a solution (0.3 ml)containing 100 mg of streptomycin sulfate at pH 7.4 in 5 mM HEPES(4-[2-Hydroxyethyl]piperazino 2-ethane sulfonic acid)/0.0725 MNaCl/0.0725 M KCl was pipetted into the glass vessel containing thediethyl ether solution of lipid. The mixture was placed in a bathsonicator (Laboratory Supplies Co., Inc.) type 10536 for severalminutes, (80 kH_(z) frequency:output 80 watts) while being dried to aviscous paste by passing thereover a gentle stream of nitrogen.

To the viscous paste remaining was added 10 ml of 5 mM HEPES. Theresulting SPLV preparation, containing streptomycin, was suspended inthe buffer solution, shaken for several minutes on a vortex mixer, andfreed of non-encapsulated streptomycin by centrifuging at 12,000 ×g for10 minutes at 20° C. The resulting cake was suspended in 0.5 ml of 5 mMHEPES.

The procedure described above was followed except that streptomycin wassubstituted by each one of the following: dihydrostreptomycin,gentamycin sulfate, ampicillin, tetracyline hydrochloride, andkanamycin.

6.2. SPLVs Containing Other Membrane Constituents

The process described in Section 6.1. was followed except that any oneof the following was added with the egg lecithin: (1) phosphatidic acidto give a molar ratio of 8:2 (lecithin:dicetylphosphate); (2)stearylamine to give a molar ratio of 8:2 (lecithin: stearylamine);cholesterol and stearylamine to give a molar ratio of 7:2:1(lecithin:cholesterol:stearylamine); and (3) phosphatidic acid andcholesterol to give a molar ratio of 7:2:1 (lecithin:phosphatidicacid:cholesterol).

6.3. SPLVs Containing Pilocarpine

The procedure of Section 6.1. was followed except that the antibioticstreptomycin was replaced with pilocarpine.

6.4. SPLVs Prepared With and Without BHT

Undistilled ether contains an anti-oxidant, 1 μg/ml butylhydroxytoluene(BHT), for storage purposes. The procedure described in Section 6.1. wasfollowing using undistilled ether as the solvent in order to incorporateBHT into the SPLV preparation.

In order to prepare SPLVs without incorporation of BHT, the proceduredescribed in Section 6.1. was followed using distilled ether as thesolvent.

7. EXAMPLE: SPLV MEDIATED DELIVERY IN VITRO

In the following example, SPLV mediated delivery of antibiotics tomacrophages in culture was demonstrated.

Peritoneal macrophages were obtained by peritoneal lavage from C₅₇ BLKadult male mice and suspended in minimal essential medium (M.E.M.) pH7.2 containing 10% heat-inactivated fetal calf serum. Cells weresuspended at a concentration of 1×10⁶ cells per ml in 96-well tissueculture dishes. To cultures containing adherent peritoneal macrophages,were added B. canis at concentrations of 1×10⁶ CFU (colony formingunits) per ml. After 12 hours, bacteria not engulfed by peritonealmacrophages were removed by repeated washings with M.E.M. After washingof peritoneal macrophage cultures, they were divided into 5 groups, eachcontaining 12 replicate cultures per group. Group 1, designatedControls, received no treatment. Group 2 received aqueous streptomycinsulfate at a concentration of 1 mg/ml. Group 3 received buffer-filledSPLVs. Group 4 received aqueous streptomycin sulfate (1 mg/ml) pluspreformed buffer-filled SPLVs. Group 5 received SPLVs containingstreptomycin sulfate (1 mg/ml). After 24 hours, supernatants wereremoved by repeated washings and peritoneal macrophages were disruptedby repeated freezing and thawing. Serial dilutions of disruptedmacrophages were plated onto brucella agar and, after 4 days, survivingB. canis were determined by limiting dilution techniques. Results shownin Table VII indicate that SPLV-entrapped streptomycin was totallyeffective in killing and eliminating the intracellular B. canisinfection in vitro.

The experiment was repeated with B. abortus exactly as described aboveexcept that peritoneal macrophages were obtained by peritoneal lavagefrom adult female albino guinea pigs. Results are also shown in TableVII.

                  TABLE VII                                                       ______________________________________                                        COLONY-FORMING UNITS OF INTRACELLULAR                                         BRUCELLA ISOLATED AFTER TREATMENT OF                                          INFECTED MACROPHAGES WITH SPLVS CONTAINING                                    STREPTOMYCIN                                                                             B. canis.sup.a                                                                            B. abortus.sup.b                                       ______________________________________                                        Controls      2.6 ± 1.13 × 10.sup.3                                                            3.1 ± 0.81 × 10.sup.4                      Buffer-filled                                                                              2.82 ± 0.10 × 10.sup.3                                                            2.9 ± 0.17 × 10.sup.4                      SPLVs                                                                         Free         3.11 + 0.40 × 10.sup.3                                                               3.3 ± 0.25 × 10.sup.4                      Streptomycin.sup.c                                                            Streptomycin 2.76 ± 0.20 × 10.sup.3                                                            2.8 ± 0.42 × 10.sup.4                      Plus Buffer-                                                                  filled SPLVs.sup.c                                                            SPLV-Entrapped                                                                             0            0                                                   Streptomycin.sup.c                                                            ______________________________________                                         .sup.a Colony forming units (CFU) of B. canis (mean ± SD of 12             replicates) isolated from equal numbers of previously infected mouse          (C.sub.57 Blk) peritoneal macrophages.                                        .sup.b CFU of B. abortus (mean ± SD of 12 replicates) isolated from        equal numbers of previously infected guinea pig peritoneal macrophages.       .sup.c Concentration of streptomycin 1 mg/ml.                            

8. EXAMPLE: TREATMENT INTRACELLULAR INFECTIONS

The following examples demonstrate how SPLVs can be used in treatingintracellular infections. The data presented demonstrates: (1) theeffectiveness of using antibiotics encapsulated in SPLVs in thetreatment of disease and (2) the greater efficiency which is obtained byadministering multiple doses of the SPLV preparation. A comparison ofMLVs to SPLVs as vehicles used in the protocols is described.

Brucellosis causes worldwide economic and public health problems.Brucellosis is caused by Brucella spp. It is adapted to many mammalianspecies, including man, domestic animals and a variety of wild animals.Six Brucella spp. cause brucellosis in animals; they are B. abortus, B.canis, B. melitensis, B. neotomae, B. ovis and B. suis. Both domesticand wild animals serve as reservoirs for potential spread of brucellosisto other animals and man.

Such infections cannot be cleared with antibiotics because theinfectious organisms reside within the cells of the reticuloendothelialsystem and are highly resistant to bactericidal activities ofantibiotics. The quantity of antibiotics required and the length oftreatment results in either toxic effects on the animal or anunacceptably high concentration of the antibiotic in the tissues of theanimal. The further difficulty in treating this disease is that thetreatment has to be completely effective since any remaining infectionwill simply spread and the cycle commences once again. The economicimpact of such diseases is demonstrated by the millions of dollars ofvaluable cattle which are lost each year due to spontaneous abortion.The only potential way to combat such infectious outbreaks is toquarantine and then slaughter the animals.

The examples which follow comprise incorporating an antibiotic intoSPLVs, and then administrating the encapsulated active substance to theanimals by inoculating the infected animals intraperitoneally.

8.1. Effect of a Single Treatment of B. Canis Infection UsingSPLV-Entrapped Antibiotic

Eighty adult male Swiss mice were infected intraperitoneally (I.P.) withB. canis ATCC 23365 (1×10⁷ CFU) and divided into 8 groups of 10 miceeach. Seven days post-inoculation with B. canis, groups were treated asfollows: Group 1, designated Controls, received no treatment; Group 2received buffer-filled SPLVs (0.2 ml I.P.); Group 3 received aqueousstreptomycin sulfate (1 mg/kg body weight in a total administration of0.2 ml I.P.); Group 4 received aqueous streptomycin sulfate (5 mg/kgbody weight) in a total administration of 0.2 ml I.P.; Group 5 receivedaqueous streptomycin sulfate (10 mg/kg body weight) in a totaladministration of 0.2 ml I.P.; Group 6 received SPLVs containingstreptomycin sulfate (1 mg/kg body weight) in a total administration of0.2 ml I.P.; Group 7 received SPLVs containing streptomycin sulfate (5mg/kg body weight) in a total administration of 0.2 ml I.P.; and Group 8received SPLVs containing streptomycin sulfate (10 mg/kg body weight) ina total administration of 0.2 ml I.P.. On day 14 post-inoculation withB. canis, all animals were sacrificed and spleens were removedaseptically. Spleens were homogenized and serially diluted onto brucellaagar to determine the number of surviving B. canis in spleens aftertreatment. Results after 4 days incubation are shown in Table VIII.

                  TABLE VIII                                                      ______________________________________                                        EFFECT OF A SINGLE TREATMENT.sup.a OF B. CANIS                                INFECTED MICE WITH VARIOUS CONCENTRATIONS                                     OF FREE OR SPLV-ENTRAPPED STREPTOMYCIN                                        ______________________________________                                                Colony-Forming Units B. Canis Per Spleen                                        No Treatment  Buffer-Filled SPLVs.sup.b                             ______________________________________                                        Control   3.46 × 10.sup.6 ±                                                                  4.1 × 10.sup.6 ± 0.66 × 10.sup.6                 2.7 × 10.sup.6                                                ______________________________________                                        Streptomycin                                                                  Concentration                                                                 (mg/kg body                                                                             Free          SPLV-Entrapped                                        weight)   Streptomycin  Streptomycin                                          ______________________________________                                        1          1.5 ± 0.45 × 10.sup.6                                                             1.01 ± 0.25 × 10.sup.3                       5         2.12 ± 1.71 × 10.sup.5                                                             1.52 ± 0.131 × 10.sup.4                      10        9.66 ± 3.68 × 10.sup.4                                                             1.32 ± 1.00 × 10.sup.4                       ______________________________________                                         .sup.a I.P. injection in total of 0.2 ml (sterile saline).                    .sup.b Surviving B. canis was determined as the number of CFU isolated pe     spleen and is expressed as mean ± S.D. of 10 animals per experiment        (triplicate experiments).                                                

8.2. Effect of Multiple Treatment of B. Canis Infection UsingSPLV-Entrapped Antibiotic

Eighty adult male Swiss mice were infected with B. canis ATCC 23365(1×10⁷ CFU, I.P.) and divided into 8 groups of 10 mice each. Seven and10 days post-inoculation with B. canis, groups were treated as follows:Group 1, designated controls, received no treatment; Group 2 receivedbuffer-filled SPLVs (0.2 ml, I.P.); Group 3 received aqueousstreptomycin sulfate (1 mg/kg body weight) in a total administration of0.2 ml, I.P.); Group 4 received aqueous streptomycin sulfate (5 mg/kgbody weight) in a total administration of 0.2 ml, I.P.; Group 5 receivedaqueous streptomycin sulfate (10 mg/kg body weight) in a totaladministration of 0.2 ml, I.P.; Group 6 received SPLVs containingstreptomycin sulfate (1 mg/kg body weight) in a total administration of0.2 ml, I.P.; Group 7 received SPLVs containing streptomycin sulfate (5mg/kg body weight) in a total administration of 0.2 ml, I.P.; and Group8 received SPLVs containing streptomycin sulfate (10 mg/kg body weight)in a total administration of 0.2 ml, I.P. On day 14 post-inoculationwith B. canis, all animals were sacrificed and spleens were removedaseptically. Spleens were homogenized and serially diluted onto brucellaagar to determine the number of surviving B. canis in spleens aftertreatment. Results after 4 days incubation are shown in FIG. 5.

The results of various two-stage treatment regimens on B. canisinfections in vivo presented in FIG. 5, demonstrate that in groupsreceiving aqueous streptomycin 7 and 10 days post-inoculation, verylittle reduction in surviving B. canis in spleens was observed. Only ingroups receiving SPLV-entrapped streptomycin at a concentration of 10mg/kg body weight administered on day 7 and 10 post-inoculation were allviable bacterial eliminated from spleens of infected animals.

In addition to the experiment described above, various tissues from B.canis infected mice after two treatments with SPLV-entrappedstreptomycin were sampled as follows:

Thirty adult male Swiss mice were inoculated with B. canis ATCC 23365(1×10⁷ CFU, I.P.). Seven days post-inoculation animals were divided into3 groups of 10 mice each. Group 1, designated controls, received notreatment; Group 2 received (on days 7 and 10 post-inoculation) aqueousstreptomycin sulfate (10 mg/kg body weight) in each administration of0.2 ml), I.P.; Group 3 received (on days 7 and 10 post-inoculation)SPLVs containing streptomycin sulfate (10 mg/kg body weight) in eachadministration of 0.2 ml, I.P. On days 14 to 75 post-inoculation with B.canis, all animals were sacrificed and the following organs removedaseptically, homogenized and serially diluted onto brucella agar forisolation of B. canis: heart, lungs, spleen, liver, kidneys, testes.After 4 days incubation, results of surviving B. canis per organ areshown in FIG. 6.

Results of samplings of various tissues in B. canis infected mice aftertwo treatment regimens with streptomycin presented in FIG. 6,demonstrated that in animals treated with SPLV-entrapped streptomycin,all tissues sampled from 14 to 75 days post-inoculation with B. caniswere totally free of any viable B. canis organisms. In animals untreatedor treated with aqueous streptomycin in concentrations andadministration schedules identical to those receiving SPLV-entrappedstreptomycin, viable B. canis organisms could be isolated in all tissuessampled from 14 to 75 days post-inoculation with B. canis.

b 8.3. Effectiveness of Treatments Using MLVs as Compared to SPLVs

Fifteen adult male Swiss mice were inoculated with B. canis ATCC 23365(1×10⁷ CFU, I.P.). Seven days post-inoculation animals were divided into3 groups of 5 mice each. Group 1, designated Controls, received notreatment; Group 2 received (on days 7 and 10 post-inoculation) MLVscontaining streptomycin sulfate (10 mg/kg body weight, I.P.). MLVs wereprepared by conventional techniques using 100 mg egg phosphatidylcholine(EPC) and 2 ml of sterile HEPES containing streptomycin sulfate (100mg/ml). The lipid to streptomycin sulfate ratio was 100 mg EPC to 28 mgstreptomycin sulfate in the 2 ml final MLV suspension; Group 3 received(on days 7 and 10 post-inoculation) SPLVs containing streptomycinsulfate (10 mg/kg body weight, I.P.) prepared as described in Section6.1. with the following modifications: 100 mg EPC were used, and 0.3 mlof HEPES containing 100 mg streptomycin sulfate. The lipid tostreptomycin sulfate ratio in SPLVs was 100 mg EPC to 28 mg streptomycinsulfate in a 2 ml final suspension. On day 14 post-inoculation with B.canis, all animals were sacrificed and spleens were removed aseptically,homogenized and serially diluted onto brucella agar for isolation of B.canis. Results of surviving B. canis per organ after 4 days incubationare shown in Table IX.

8.4. Effect of Various SPLV-Entrapped Antibiotics on Treatment ofInfection

Fifty adult male Swiss mice were inoculated with B. canis ATCC 23365(1×10⁷ CFU, I.P.). Seven days post-inoculation, animals were dividedinto 10 groups of 5 mice each. Group 1, designated controls, received notreatment; Group 2 received buffer-filled SPLVs (0.2 ml, I.P.) on days 7and 10 post-inoculation; Groups 3, 4, 5 and 6 received aqueousinjections (0.2 ml I.P.) of dihydrostreptomycin, gentamycin, kanamycinor streptomycin 10 mg/kg body weight, I.P. on days 7 and 10post-inoculation (N.B. Each of these antibiotics have been shown to killB. canis in vitro).

                  TABLE IX                                                        ______________________________________                                        COMPARISON OF MLVS AND SPLVS CONTAINING                                       STREPTOMYCIN SULFATE ON KILLING OF                                            B. CANIS IN VIVO AFTER TWO TREATMENTS.sup.a                                             Colony-Forming Units                                                          B. Canis per Spleen.sup.b                                           ______________________________________                                        Control     2.7 ± 1.0 × 10.sup.4                                     MLVs.sup.c  1.8 ± 0.4 × 10.sup.4                                     SPLVs.sup.c 0                                                                 ______________________________________                                         .sup.a Intraperitoneal injections, 10 mg/kg body weight, were spaced at 3     day intervals. Controls received no treatment.                                .sup.b Surviving B. canis was determined as the number of CFU isolated pe     spleen and is expressed as the mean ± S.D. of 5 animals per group          (duplicate determinations per animal).                                        .sup.c Egg phosphatidylcholine to streptomycin sulfate ratios were 100 mg     lipid to 28 mg streptomycin sulfate.                                     

Groups 7, 8, 9, and 10 received SPLVs containing dihydrostreptomycin,gentamicin, kanamycin, or streptomycin at 10 mg/kg body weight on days 7and 10 postinoculation. On day 14 post-inoculation with B. canis, allanimals were sacrificed and spleens were removed aseptically,homogenized and serially diluted onto brucella agar for at isolation ofB. canis. Results of surviving B. canis per organ after 4 daysincubation are as shown in Table X.

                  TABLE X                                                         ______________________________________                                        COMPARISON OF VARIOUS ANTIBIOTICS ON KILLING                                  OF B. CANIS IN VIVO AFTER TWO TREATMENTS.sup.a                                           Colony-Forming Units B. Canis Per Spleen.sup.b                                Aqueous     SPLV-Entrapped                                                    Solutions   Antibiotic                                             ______________________________________                                        Untreated    3.93 ± 1.51 × 10.sup.6                                                             4.66 ± 0.87 × 10.sup.6                    Antibiotic.sup.c                                                              Dihydrostreptomycin                                                                        1.13 ± 0.30 × 10.sup.5                                                             0                                                  Gentamycin   7.06 ± 2.53 × 10.sup.5                                                             0                                                  Kanamycin    2.72 ± 0.91 × 10.sup.5                                                             0                                                  Streptomycin 1.01 ± 0.17 × 10.sup.5                                                             0                                                  ______________________________________                                         .sup.a Intraperitoneal treatments, 10 mg/kg body weight, were spaced at 3     day intervals. Controls received no treatment.                                .sup.b Surviving B. canis per organ was determined as the number of CFU       isolated per spleen and expressed as the mean ± S.D. of 5 animals per      groups (duplicate determinations per animal).                                 .sup.c Antibiotics effective in killing B. canis in suspension culture.  

The results from tests of various antibiotics on B. canis infected micepresented in Table X demonstrate that antibiotics which are effective inkilling B. canis in vitro (i.e., in suspension culture) are also onlyeffective in killing B. canis infections in vivo when they areencapsulated within SPLVs. Animals receiving either aqueous antibiotics,buffer-filled SPLVs or no treatment were in no case cleared of survivingB. canis in isolated spleen tissues.

8.5 Treatment of Dogs Infected with B. canis

Adult female beagles were inoculated with B. canis ATCC 23365 (1×10⁷CFU) orally and vaginally. Seven days post-inoculation dogs were dividedinto 3 groups. Group 1, designated control, received no treatment; Group2 received (on days 7 and 10 post-inoculation) aqueous streptomycinsulfate at 10 mg/kg body weight (each administration was 5.0 ml, I.P.).Group 3 received (on days 7 and 10 post-inoculation) SPLVs containingstreptomycin sulfate at 10 mg/kg body weight (each administration was3.0 ml, I.P.). Vaginal swabbings of dogs and heparinized blood sampleswere collected at regular intervals before, during, and at thetermination of the study. These were cultured on brucella agar in orderto isolate B. canis. Results are shown in Table XI. Serum samples werecollected before, during, and at the termination of the study fordeterminations of serum antibody against B. canis. These results arealso shown in Table XI. Twenty-one days post-inoculation with B. canis,all animals were euthanized. The following tissues were removedaseptically, homogenized and serially diluted onto brucella agar forisolation of B. canis: heparinized blood, vaginal exudate, lungs,spleen, synovial fluid, uterus, ovary, popliteal lymph nodes, salivaryglands, tonsils, mediastinal lymph nodes, mesenteric lymph nodes, bonemarrow, superficial cervical lymph nodes, and auxiliary lymph nodes.Results of surving B. canis per tissue after 4 days incubation are shownin Table XII.

                                      TABLE XI                                    __________________________________________________________________________    RESULTS OF CULTURES AND SEROLOGICAL TESTING                                   IN B. CANIS INFECTED DOGS SUBJECTED                                           TO A TWO TREATMENT ANTIBIOTIC REGIMEN.sup.a                                   Days After                   SPLV-                                            Infection                    Entrapped                                        with     Control   Streptomycin.sup.b                                                                      Streptomycin.sup.c                               B. Canis R  M  B V R  M  B V R  M  B V                                        __________________________________________________________________________    Pre-treatment                                                                 0        0  0  0 0 0  0  0 0 0  0  0 0                                        2        ND ND + + ND ND + 0 ND ND + +                                        4        ND ND + + ND ND + + ND ND + +                                        Post-treatment                                                                8        0  0  0 + 0  0  + 0 0  0  0 0                                        10       0  0  0 + 0  0  0 + 0  0  0 0                                        21       1.5                                                                              2  + + 1  2  + + 0  0  0 0                                        __________________________________________________________________________     .sup.a R (rapid slide agglutination test) indicates the reciprocal of         serum titer to B. canis antigen (× 10.sup.2); 0 = no detectable         titer.                                                                        M (2mercaptoethanol tube agglutination test) indicates the reciprocal of      serum titer to B. canis antigen (× 10.sup.2); 0 = no detectable         titer.                                                                        In B (blood culture) and V (vaginal culture) on brucella agar: + =            detection of greater than or equal to 1 CFU; 0 = no colonies detected.        Controls received no treatment.                                               .sup.b Streptomycin sulfate (aqueous) 10 mg/kg body weight, I.P.              .sup.c SPLVs containing streptomycin sulfate 10 mg/kg body weight, I.P.  

                  TABLE XII                                                       ______________________________________                                        RESULTS OF CULTURES FROM TISSUE SAMPLES                                       IN B. CANIS INFECTED DOGS SUBJECTED                                           TO A TWO TREATMENT ANTIBIOTIC REGIMEN.sup.a                                                 SPLVs                                                                         Containing              Con-                                    Tissue.sup.b  Streptomycin.sup.c                                                                        Streptomycin.sup.d                                                                        trol.sup.e                              ______________________________________                                        Whole blood   0           +           +                                       Vaginal swab  0           +           +                                       Lungs         0           +           +                                       Spleen        0           +           +                                       Synovial fluid                                                                              N.D.        0           0                                       Uterus        0           +           +                                       Ovary         0           +           +                                       Popliteal lymph node                                                                        N.D.        +           +                                       Salivary gland                                                                              0           0           0                                       Tonsil        0           +           +                                       Mediastinal lymph node                                                                      0           N.D.        +                                       Mesenteric lymph node                                                                       N.D.        0           0                                       Bone marrow   0           +           +                                       Superficial   N.D.        N.D.        +                                       cervical lymph node                                                           Axillary lymph node                                                                         0           +           +                                       ______________________________________                                         .sup.a Animals treated on day 7 and 10 postinfection.                         .sup.b Samples taken at necropsy were serially diluted on brucella agar;      = equal to or greater than 1 CFU; 0 = no colonies.                            .sup.c SPLVs containing streptomycin sulfate, 10 mg/kg body weight, I.P.      .sup.d Streptomycin sulfate (aqueous), 10 mg/kg body weight, I.P.             .sup.e Controls received no treatment.                                   

Results of culture and serologic tests of dogs infected with B. canisbefore, during, and after two-stage antibiotic administration arepresented in Table XI. All animals were serologically negative forprevious exposure to B. canis as measured by negative serum titers, andwere culture negative from blood cultures and cultures of vaginalswabbings. All animals were noted to be culture positive for both bloodand vaginal cultures prior to treatments on days 7 and 10. Dogs treatedwith aqueous streptomycin or dogs receiving no treatment remainedculture positive for blood and vaginal cultures during post-treatmentperiods prior to termination on day 21. Group 3, which receivedliposomes containing streptomycin, became culture negative one day afterthe first treatment and remained negative throughout post-treatmentperiod. Dogs which received no treatment or aqueous streptomycindeveloped detectable serum titers against B. canis antigens by day 21post-inoculation, while those treated with SPLVs containing antibioticson days 7 and 10 post-inoculation did not develop any detectableantibody to B. canis antigen.

Results from isolation of B. canis from infected dogs treated withtwo-stage antibiotic administration which are presented in Table XIIdemonstrate that in dogs, only treatment with SPLVs containingstreptomycin was effective in eliminating any viable B. canis in alltissues from all organ samples.

8.6. Treatment of B. Abortus in Guinea Pigs

Fifteen adult female guinea pigs were inoculated with B. abortus ATCC23451 (1×10⁷ CFU, I.P.). Seven days post-inoculation animals weredivided into 3 groups of 5 animals each. Group 1, designated Controls,received no treatment. Group 2 received aqueous streptomycin sulfate,I.P. injections (0.2 ml) at 10 mg/kg body weight on day 7 and 10post-inoculation with B. abortus. Group 3 received SPLVs containingstreptomycin sulfate I.P. injections (0.2 ml) at 10 mg/kg body weight ondays 7 and 10 post-inoculation with B. abortus. On day 14post-inoculation with B. abortus, all animals were sacrificed andspleens were removed, aseptically homogenized and serially diluted ontobrucella agar for isolation of B. abortus. Results of surviving B.abortus per spleen after 4 days incubation, are shown in FIG. 7. OnlySPLVs containing streptomycin were effective in eliminating B. abortusresiding within guinea pig spleen. In animals receiving aqueousstreptomycin or no treatment, viable B. abortus bacteria were beidentified.

8.7. Treatment of B. abortus Infection in Cows

Nine heavily infected animals were utilized in this experiment. B.abortus bacterial isolations from milk and vaginal swabbings became andremained negative for six weeks following treatment with SPLVscontaining streptomycin. When infection reoccurred in these animals,bacterial isolations were found only in quadrants of the udder whichwere positive prior to treatment.

Nine cross-bred (hereford-jersey-Brangus), 22-month old, non-gravid,confirmed B. abortus culture-positive cows were used. At least 4 monthsprior to the initiation of the study, the animals were experimentallychallenged per conjunctivum with 1×10⁷ CFU of B. abortus Strain 2308during mid-gestation, which resulted in abortion and/or B. abortusculture positive lacteal or uterine secretions and/or fetal tissues.

Cows were maintained in individual isolation stalls and separated intothree groups. Treatment comprised a two-dose regimen, spaced 3 daysapart, as follows: (1) 3 cows were injected intraperitoneally withphysiological saline. (2) 3 cows were injected intraperitoneally withaqueous antibiotic (streptomycin at 10 mg/kg body weight) plus preformedbuffer-filled SPLVs. (3) 3 cows were injected intraperitoneally withSPLV-entrapped streptomycin (10 mg/kg body weight). The total volume perinjection was 100 ml per animal.

During the first 2 months duplicate bacteriologic cultures of lactealand uterine secretions were performed weekly providing secretions wereobtainable. Then, all 5 cows were euthanatized with an overdose ofsodium pentabarbitol, and the following organs were collected induplicate for bacteriologic cultures: (1) lymph nodes: left and rightatlantal, left and right suprapharyngeal, left and right mandibular,left and right parotid, left and right prescapular, left and rightprefemoral, left and right axillary, left and right popliteal, left andright internal iliac, left and right supramammary, left and right renal,bronchial, mediastinal, mesenteric, and hepatic; (2) glands: all fourquarters of mammary gland, left and right adrenal glands and thymus (ifpresent); (3) organs and other tissues: spleen, liver, left and righthorn of uterus, cervix, vagina, kidney and tonsil.

After necropsy, all tissues were frozen and maintained at -70° C. whilein transport. Tissues were thawed, alcohol flamed, and asepticallytrimmed prior to weighing. Once weights were recorded (0.2 to 1.0grams), the tissue was homogenized in 1 ml of sterile saline andserially diluted with sterile saline to 1:10⁻¹⁰ of initial homogenatesuspension. Aliquots (20 μl) of each dilution from serial suspensionswere plated onto brucella agar and placed in 37° C. incubation.Duplicate determinations were performed for each tissue.

Plates were read daily and scored for bacterial growth. All coloniesappearing prior to 3 days were isolated, passaged, and gram stained todetermine identity. On days 5, 6 and 7 during incubation colonies withmorphology, growth, and gram staining characteristics consistent with B.abortus were counted; the CFU per gram tissue was then determined.Representative colonies were repassaged for bacterial confirmation of B.abortus.

Bacteriologic isolations were done on all tissue samples andquantitation of bacteria per gram of tissue were calculated. The resultsfrom four animals--one placebo control and three animals treated withSPLV-entrapped streptomycin--are presented in Table XIII.

EXAMPLE: TREATMENT OF OCULAR AFFLICTIONS

Bacterial and like infections as well as many other afflictions of theeye cause worldwide economic and public health problems, leading, ifuntreated or improperly treated, to loss of sight and possible death dueto septicemia. Bacterial infections of the eye in animals and man havebeen reported to be caused by a variety of bacteria including but notlimited to: Clostridium spp., Corynebacterium spp., Leptospira spp.,Moraxella spp., Mycobacterium spp., Neisseria spp., Propionibacteriumspp., Proteus spp., Pseudomonas spp., Serratia spps., E. Coli spp.,Staphylococcus spp., Streptococcus spp. and bacteria-like organismsincluding Mycoplasma spp. and Rickettsia spp. Both animals and man serveas reservoirs for potential spread of infectious bacteria to each other.

                  TABLE XIII                                                      ______________________________________                                        RESULTS OF CULTURES FROM TISSUE                                               SAMPLES OF B. ABORTUS INFECTED COWS                                                                  SPLV-Entrapped                                                        Untreated                                                                             Streptomycin                                           Tissue           Control   1      2    3                                      ______________________________________                                        Adrenal gland L  0         0      0    0                                      Adrenal gland R  ++        0      0    +                                      Atlantal LN R    ++        +      0    +                                      Atlantal LN L    0         0      0    +                                      Axillary LN R    +++       0      +    0                                      Axillary LN L    ++        0      0    0                                      Bronchial LN     0         0      0    0                                      Cervix           0         0      0    0                                      Hepatic LN       ++++      0      0    0                                      Horn of Uterus L 0         0      0    +                                      Horn of Uterus R 0         0      0    0                                      Int. Illiac LN R ++        0      0    0                                      Int. Illiac LN L ++++      0      +    0                                      Kidney           0         0      0    0                                      Liver            0         0      0    0                                      Lung             0         0      0    0                                      Mammary Gland LF 0         +      +    0                                      Mammary Gland LR 0         0      0    +                                      Mammary Gland RF + +       0      0    0                                      Mammary Gland RR ++        0      0    0                                      Mandibular LN R  +++       0      0    0                                      Mandibular LN L  +++       0      0    0                                      Mediastinal LN   ++        0      +    0                                      Mesenteric LN    +++       0      0    0                                      Parotid LN L     +++       0      0    0                                      Parotid LN R     +++       0      0    0                                      Popliteal LN L   +         0      0    0                                      Popliteal LN R   +         0      0    0                                      Prefemoral LN L  +         0      0    0                                      Prefemoral LN R  0         0      0    0                                      Prescapular LN L 0         0      0    +                                      Prescapular LN R ++++      0      0    0                                      Renal LN         0         0      0    0                                      Spleen           +++       0      0    0                                      Supramammary LN L                                                                              +++       +      0    0                                      Supramammary LN R                                                                              0         0      0    0                                      Suprapharangeal LN L                                                                           +         0      0    0                                      Suprapharangeal LN R                                                                           0         0      0    0                                      Thymus           0         0      0    0                                      Vagina           +++       0      0    0                                      ______________________________________                                         0 No detectable bacteria by culture of 0.3-1 gm of tissue.                    + Less than 200 colonies/gm tissue.                                           ++ More than 300 colonies/gm.                                                 +++ More than 1,000 colonies/gm.                                              ++++ More than 100,000 colonies/gm.                                      

Such bacterial infections cannot be treated with antibiotics withoutlengthy and cumbersome treatment schedules resulting in either frequenttreatments, as rapid as every twenty minutes in humans with someinfections, or unacceptably high concentrations of the antibiotic in thetissues. Current treatment methods are difficult for many other reasons.The infectious organism in the surface tissues of the eye in some casesare highly resistant to bactericidal activities of antibiotics, andtopical administration of antibiotics can result in rapid clearing ofthe drug from the eye socket yielding varying contact times. As ageneral rule, treatment of eye infections has to be completely effectivesince any remaining infection will simply reinfect through lacrimalsecretions and the cycle commences once again. Further, in many casesdrug concentrations needed to eliminate the infection can causeimpairment of vision and in certain cases can result in total blindness.The economic impact of such diseases in domestic animals is demonstratedby the millions of dollars which are lost each year since the onlypotential way to combat such infectious diseases is sustained therapyand quarantine.

The following experiments evaluate the effectiveness of treatments usingfree antibiotic in glycerine as compared to antibiotic entrapped inSPLVs for M. bovis infections of the eye.

M. bovis causes infectious keratoconjunctivitis (pink-eye) in cattle.This condition is characterized by blepharospasm, lacrimation,conjunctivitis and varying degrees of corneal opacity and ulceration.Adult cows may develop a mild fever with slightly decreased appetite anda decreased milk production. Although a number of antibiotics areeffective against M. bovis, they must be administered early and repeatedoften by topical application or subconjuctival injection.

According to the examples described herein, the effectiveness andduration of action of the therapeutic substance are prolonged. It issurprising that this sytem is effective with only one or twoadministrations since such infections do not respond to simple ordinarytreatment with antibiotics. The usual treatments often leave smallremaining infections which reinfect the eye so that the infectious cyclewill commence again, unless the infection is completely eradicated bynumerous repetitions of the treatment.

9.1. Treatment of Infectious Keratoconjunctivitis in Mice

C57 black mice (160 mice) were divided into 8 groups. One half of eachgroup was exposed to U.V. irradiation in each eye (in order to createcorneal lesions). All animals were then inoculated with M. bovisinstilled onto the right eye at concentrations of 1×10⁶ bacteria pereye. Twenty-four hours post-inoculation all animals were scored fordegree of corneal opacity. The eight groups were treated by topicalapplication of the following to each eye: Groups 1 and 2 received 10 μlof SPLV-entrapped streptomycin (30 mg/ml); Groups 3 and 4 received 10 μlstreptomycin (100 mg/ml); Groups 5 and 6 received 10 μl of buffer-filledSPLVs suspended in aqueous streptomycin (100 mg/ml); and Groups 7 and 8received 10 μl of sterile saline (N.B. The uninfected left eyes weretreated with the same topical solutions in order to determine whetherSPLVs would irritate the eye; no irritation was observed). Once daily,animals were scored for progression or regression of corneal lesions andon days 3, 5 and 7 post-treatment right eyes were swabbed and isolationsfor M. bovis were performed on representative animals. M. bovis colonieswere determined by colony morphology and reactivity to fluorescentlylabeled antibody to M. bovis pili. Results, shown in Table XIV, revealthat only the SPLV-entrapped streptomycin was effective in eliminatinginfection.

9.2 Treatment of Rabbit Conjunctiva Using SPLV-Entrapped Antibiotic

M. bovis, ATCC strain 10900, were diluted to a concentration of 1×10⁷cells per ml in sterile saline (0.085% NaCl). Aliquots (0.1 ml) ofbacterial suspensions were inoculated topically into the eyes of tenadult female rabbits. Samples for cultures were taken daily by swabbingthe conjunctivae and plated onto blood agar plates for isolation of M.bovis. Three days post-inoculation, rabbits were divided into 3 groups:2 animals (controls) received no treatment; 4 animals receivedstreptomycin in sterile saline (concentration 10 mg/kg body weight); and4 animals received SPLV-entrapped streptomycin in a sterile salinesolution (concentration 10 mg streptomycin/kg body weight). Allsolutions were administered topically into each eye. After 24 hours, theswabbing of conjunctivae of all rabbits was resumed and continued dailyfor seven days. The results of isolation for M. bovis on blood agarplates are shown in Table XV.

9.3. Treatment of Keratoconjunctivitis Resulting from SubcutaneousInfections

M. bovis ATCC strain 10900, were diluted to a concentration of 1×10⁷cells per ml in sterile saline. Aliquots (0.1 ml) of bacterialsuspensions were inoculated into the eyes of adult rabbits which hadbeen previously infected as described in Section 9.2. and were nottreated with SPLVs.

                                      TABLE XIV                                   __________________________________________________________________________    RESULTS OF TREATMENT OF INFECTIOUS                                            KERATOCONJUNCTIVITIS RESULTING FROM                                           OCULAR INFECTIONS OF M. BOVIS IN MICE                                                                         M. Bovis                                                 Number of Mice Per Group of 20                                                                     Cultures.sup.a                                           Pre-Treatment                                                                            Post-Treatment                                                                          Days Post-                                               Corneal Opacity.sup.b                                                                    Corneal Opacity.sup.b                                                                   Treatment                                                0 1 2 3  4 0 1 2 3 4 3  5                                          __________________________________________________________________________    Non-radiated Mice                                                             Controls   16                                                                              3 0 1  0 18                                                                              2 0 0 0 4/5                                                                              4/5                                        Free       18                                                                              1 1 0  0 18                                                                              2 0 0 0 2/5                                                                              2/5                                        Streptomycin.sup.c                                                            Buffer-filled                                                                            17                                                                              2 1 0  0 18                                                                              1 1 0 0 2/5                                                                              3/5                                        SPLVs plus free                                                               Streptomycin.sup.c                                                            SPLVs-Entrapped                                                                          17                                                                              3 0 0  0 20                                                                              0 0 0 0 0/5                                                                              0/5                                        Streptomycin.sup.c                                                            UV-Radiated Mice                                                              Controls    1                                                                              1 5 9  4 10                                                                              3 1 2 4 5/5                                                                              5/5                                        Free        0                                                                              4 9 7  0 14                                                                              3 2 1 0 3/5                                                                              4/5                                        Streptomycin.sup.c                                                            Buffer-filled                                                                             0                                                                              3 5 10 2 11                                                                              2 4 3 0 3/5                                                                              3/5                                        SPLVs plus free                                                               Streptomycin.sup.c                                                            SPLVs-Entrapped                                                                           0                                                                              1 5 11 3 19                                                                              1 0 0 0 0/5                                                                              0/5                                        Streptomycin.sup.c                                                            __________________________________________________________________________     .sup.a Culture of eyes positive for presence of M. bovis, determined by       fluorescent antibody staining.                                                .sup.b Scoring of normal cornea: 1 = loss of normal luster; 2 = small foc     of opacity; 3 = partial opacity of cornea; 4 = total opacity of cornea.       .sup.c Total administration 10 μl (1.0 mg streptomycin per eye).      

                  TABLE XV                                                        ______________________________________                                        RESULTS OF ISOLATION FROM RABBIT                                              CONJUNCTIVAE AFTER TOPICALLY INFECTING                                        WITH M. BOVIS AND TREATING WITH                                               AQUEOUS OR SPLV-ENCAPSULATED STREPTOMYCIN                                                   M. bovis Cultures.sup.a                                                       Days Post-Infection                                                             Pre-                                                                   Animal Treatment.sup.b                                                                          Post-Treatment.sup.c                               Group      Number   1       2    3   4   5   6   7                            ______________________________________                                        Control    1        0       +    +   +   +   +   +                                       2        0       +    +   +   +   +   +                            Streptomycin.sup.d                                                                       1        0       +    +   +   +   +   +                                       2        0       0    +   +   +   +   +                                       3        0       +    +   +   +   +   +                                       4        0       +    +   +   +   +   +                            SPLV-Entrapped                                                                           1        0       0    +   0   0   0   0                            Streptomycin.sup.e                                                                       2        0       +    +   0   0   0   0                                       3        0       +    +   0   0   0   0                                       4        0       +    +   0   0   0   0                            ______________________________________                                         .sup.a Cultures scored for presence of M. bovis colonies on blood agar        plates after 24 hours at 37° C. Plus (+) represents greater than o     equal to 1 CFU M. bovis per isolate; 0 represents no detectable colonies.     .sup.b All animals inoculated with 1 × 10.sup.6 CFU M. bovis            topically in each eye.                                                        .sup.c Animals treated with 0.1 ml solution topically in each eye.            .sup.d Streptomycin (10 mg/kg body weight) in sterile saline solution.        .sup.e SPLVentrapped streptomycin (10 mg/kg body weight) in sterile salin     solution.                                                                

The right eyes of all nine rabbits were inoculated with 0.1 ml of M.bovis subcutaneously into conjunctival tissue and in the left eyes ofall rabbits were inoculated with 0.1 ml of M. bovis topically. Cultureswere taken daily from conjunctivae of both eyes from all rabbits andplated onto blood agar plates for isolation of M. bovis. Three dayspost-inoculation, rabbits were divided into 3 groups: 2 animals receivedno treatment; 3 animals received streptomycin in a standard ophthalmicglycerin suspension (concentration of streptomycin 10 mg/kg bodyweight); and 4 animals received a saline suspension of SPLV-entrappedstreptomycin (10 mg of streptomycin sulfate per kg of body weight). Thesuspension or solution was administered topically (0.1 ml) into eacheye. After 24 hours and on each of the next five days, conjunctivalswabbings were taken from all rabbits. The results of isolation for M.bovis on blood agar plates are shown in Table XVI. Necropsies wereperformed on all animals at the termination of experiments andconjunctivae were removed from all animals. These were scored forvascularization, and were minced, homogenized and plated onto blood agarplates for isolation of M. bovis. Results are shown in Table XVII.

9.4. Evaluation of the Effectiveness of SPLVs as Compared to LiposomePreparations in the Treatment of ocular Infections

M. bovis (ATCC strain 10900) were diluted to a concentration of 1×10⁷cells per ml in sterile saline. Aliquots (0.1 ml) of bacterialsuspensions were inoculated subcutaneously into the conjunctival tissuesof both eyes in adult rabbits. Swabbings were taken daily fromconjunctivae of both eyes from all rabbits and plated onto blood agarplates for isolation of M. bovis.

                  TABLE XVI                                                       ______________________________________                                        RESULTS OF ISOLATION FROM RABBIT CONJUNC-                                     TIVAE AFTER INOCULATION OF M. BOVIS INTO                                      CONJUNCTIVAL MEMBRANES AND TREATMENT WITH                                     STREPTOMYCIN IN OPHTHALMIC                                                    GLYCERINE SOLUTION                                                            OR SPLV-ENCAPSULATED STREPTOMYCIN IN SALINE                                                  M. bovis Cultures.sup.a                                                       Days Post Infection.sup.c                                               Animal  Pre-treatment                                                                            Post-Treatment                                    Group      Number.sup.b                                                                            1      2     3    4    5                                 ______________________________________                                        Control    1         +      +     +    +    +                                            2         +      +     +    +    +                                 Streptomycin                                                                             1         +      +     +    +    +                                 in Glycerine                                                                             2         +      +     +    +    +                                 solution.sup.d                                                                           3         +      +     +    +    +                                 SPLV-      1         +      +     0    0    0                                 Encapsulated                                                                             2         +      +     0    0    0                                 Streptomycin.sup.e                                                                       3         +      +     0    0    0                                            4         +      +     0    0    0                                 ______________________________________                                         .sup.a Cultures scored for presence of M. bovis colonies on blood agar        plates after 24 hours at 37° C. Plus (+) represents greater than o     equal to 1 CFU M. bovis per isolate; 0 represents no detectable colonies.     .sup.b All animals were inoculated with 1 × 10.sup.6 CFU M. bovis       topically in both eyes; 1 × 10.sup.6 CFU M. bovis was injected into     conjunctival membranes, in right eyes; and 1 × 10.sup.6 CFU M. bovi     was applied topically in left eyes.                                           .sup.c Animals treated with 0.1 ml solution topically in each eye.            .sup.d Animals treated topically in each eye with streptomycin (10 mg/kg      body weight) in ophthalmic glycerine base.                                    .sup.e Animals treated topically in each eye with SPLVencapsulated            streptomycin (10 mg/kg body weight) in sterile saline solution.          

                  TABLE XVII                                                      ______________________________________                                        RESULTS FROM NECROPSY OF THE ORBIT AND ASSO-                                  CIATED TISSUES FROM RABBITS AFTER INOCULA-                                    TION WITH M. BOVIS INTO CONJUNCTIVAL TISSUES                                  AND TREATMENT WITH EITHER STREPTOMYCIN IN                                     OPHTHALMIC GLYCERINE SOLUTION OR SPLV-ENCAP-                                  SULATED STREPTOMYCIN IN STERILE SALINE.sup.a                                                Isolation                                                                     of M. bovis                                                                           Vascularization                                                       Cultures                                                                              of Right Eye.sup.b                                      ______________________________________                                        Control                                                                       A               +         2+                                                  B               +         2+                                                  Streptomycin in                                                               Glycerine Solution                                                            A               +         2+                                                  B               +         1+                                                  C               +         2+                                                  D               +         2+                                                  SPLV-encapsulated                                                             Streptomycin                                                                  A               0         0                                                   B               0         0                                                   C               0         0                                                   D               0         0                                                   ______________________________________                                         .sup.a Legends are same as Table XII, performed on day 5, post infection.     .sup.b Vascularization scored as follows: 0 = vessels normal; 1 = some        vessels definitely dilated and infiltrated by minor vessels; 2 = diffuse      red with individual vessels not easily discernible; 3 = diffuse beefy red     vascular leakage and effusion of blood into conjunctivae.                

Five days post-inoculation, rabbits were divided into 6 groups: 2animals received no treatment (controls); 3 animals received asuspension of SPLV-encapsulated streptomycin (10 mg of streptomycinsulfate per kg of body weight) which when diluted 1:100 had an O.D.₄₈₀(optical density at 480 nm) equal to 0.928; 3 animals received asuspension of SPLV-encapsulated streptomycin (10 mg of streptomycinsulfate per kg of body weight) which when diluted 1:100 had an O.D.₄₈₀equal to 0.449; 3 animals received a suspension of SPLV-encapsulatedstreptomycin (10 mg streptomycin sulfate per kg of body weight) whichwhen diluted 1:100 had an O.D.₄₈₀ equal to 0.242; 3 animals received asuspension of SPLV-encapsulated streptomycin (10 mg streptomycin sulfateper kg body weight) which when diluted 1:100 had an O.D.₄₈₀ equal to0.119; and 2 animals received a suspension of multilamellar vesicles(MLVs) containing streptomycin (10 mg streptomycin sulfate per kg ofbody weight) with an O.D.₄₈₀ of a 1:100 dilution equal to 0.940. MLVSwere made by the process of Fountain et al. Curr. Micro. 6:373 (1981) byadding streptomycin sulfate to the dried lipid film which was thenvortexed, and allowed to swell for two hours; the non-entrappedstreptomycin was removed by repeated centrifugation.

The suspensions were administered topically into each eye. After 24hours, conjunctival swabbings were taken from all rabbits daily for 9days and plated onto blood agar. The results of isolation for M. bovison blood agar plates are shown in Table XVIII. Necropsies were performedon all animals. These were scored for lacrimal secretions, andconjunctivae were removed aseptically from all animals. These werescored for vascularization, and were minced, homogenized and plated ontoblood agar plates for isolation of M. bovis. Results are shown in TableXIX.

10. EXAMPLE: TREATMENT OF VIRAL INFECTIONS

Lymphocytic choriomeningitis virus (LCMV), a member of the Arenavirusgroup, is known to cause diseases in man and LCMV infection is fatal inmice inoculated intracerebrally with this virus. The death of mice iscaused by the immune cells which react against virus-infected cells. Thevirus does not kill the cells in which it multiplies, therefore, thetherapeutic agent used in mice must either inhibit virus multiplicationso that the immune cells will not be activated, and/or inhibit theactivation of immune cells.

The following example demonstrates the effectiveness of treating viralinfections by administering a SPLV-encapsulated antiviral compound.

10.1. Treatment of Lethal Lymphocytic Choriomeningitis Virus Infectionsin Mice

Swiss mice 2 months of age were inoculated intracerebrally with a lethaldose of LCM virus, i.e., 100 plaque forming units (PFU) in 0.05 mlinoculum per mouse. Mice were divided into 4 groups of 7 animals eachand were treated on days 2, 3 and 4 post-infection by intraperitonealinjections with 0.1 ml/dose/mouse as follows: (1) the "SPLV-R group" wastreated with a suspension of egg phosphatidylcholine SPLVs containing 3mg Ribavarin/ml.

                                      TABLE XVIII                                 __________________________________________________________________________    ISOLATION OF M. BOVIS FROM INFECTED RABBIT                                    CONJUNCTIVAE AFTER TREATMENT WITH DILUTIONS                                   OF SPLV-ENCAPSULATED STREPTOMYCIN IN SALINE                                   OR MLV-ENCAPSULATED STREPTOMYCIN IN SALINE                                                Isolation of M. bovis.sup.a                                                   Days Post-Infection                                                      Animal                                                                             Pre-Treatment                                                                           Post-Treatment                                          Group  Number                                                                             1 2 3 4 5 6 7 8 9 10                                                                              11                                                                              12                                                                              13                                                                              14                                      __________________________________________________________________________    Control                                                                              1    + + + + + + + + + + + + + +                                              2    + + + + + + + + + + + + + +                                       MLV-   1    + + + + + + + 0 0 + + + 0 +                                       encapsulated                                                                         2    + + + + + + 0 + + 0 0 + + 0                                       Streptomycin                                                                  SPLV-  1    + + + + + 0 0 0 0 0 0 0 0 0                                       encapsulated                                                                         2    + + + + + 0 0 0 0 0 0 0 0 0                                       Streptomycin                                                                         3    + + + + + 0 0 0 0 0 0 0 0 0                                       (undiluted)                                                                   SPLV-  1    + + + + + + 0 + + + + + + +                                       encapsulated                                                                         2    + + + + + 0 0 + 0 0 0 0 0 0                                       Streptomycin                                                                         3    + + + + + 0 0 0 0 + 0 0 0 0                                       (1:2 dilution)                                                                SPLV-  1    + + + + + + + 0 0 0 0 0 0 0                                       encapsulated                                                                         2    + + + + + + 0 0 0 + 0 0 0 0                                       Streptomycin                                                                         3    + + + + + 0 0 + + + + + + +                                       (1:4 dilution)                                                                SPLV-  1    + + + + + 0 0 + + + + + + +                                       encapsulated                                                                         2    + + + + + 0 0 0 0 0 0 0 0 0                                       Streptomycin                                                                         3    + + + + + + + + 0 + + + + +                                       (1:6 dilution)                                                                __________________________________________________________________________     .sup.a All animals inoculated with 1 × 10.sup.6 CFU M. bovis by         injection into conjunctival membranes of both eyes. Conjunctival swabbing     were plated on blood agar. Cultures scored for presence of M. bovis           colonies on blood agar plates after 24 hours at 37° C.; + = greate     than or equal to 1 CFU; 0 = no detectable cultures.                      

                  TABLE XIX                                                       ______________________________________                                        RESULTS FROM NECROPSY OF THE ORBIT AND                                        ASSOCIATED TISSUES FROM RABBITS AFTER                                         INOCULATION WITH M. BOVIS INTO CONJUNCTIVAL                                   TISSUES AND TREATMENT WITH EITHER MLV-EN-                                     CAPSULATED STREPTOMYCIN, SPLV-ENCAPSU-                                        LATED STREPTOMYCIN OR DILUTIONS OF SPLV-                                      ENCAPSULATED STREPTOMYCIN.sup.a                                                             Isolation Vasculariza-                                                                             Lacrimal                                          Animal of M. Bovis                                                                             tion of Eyes.sup.b                                                                       Discharge.sup.c                            ______________________________________                                        Control  1        +         1+        1+                                               2        +         1+        1+                                      MLV-     1        +         1+        1+                                      encapsulated                                                                           2        0         1+       0                                        Streptomycin                                                                  SPLV-    1        0         0        0                                        encapsulated                                                                           2        0         1+       0                                        Streptomycin                                                                           3        0         0        0                                        (undiluted)                                                                   SPLV-    1        +         2+        2+                                      encapsulated                                                                           2        0         0        0                                        Streptomycin                                                                           3        0         1+       0                                        (1:2 dilution)                                                                SPLV-    1        0         0        0                                        encapsulated                                                                           2        0         1+       0                                        Streptomycin                                                                           3        +         1+       0                                        (1:4 dilution)                                                                SPLV-    1        +         1+        1+                                      encapsulated                                                                           2        0         1+       0                                        Streptomycin                                                                           3        +         1+       0                                        (1:6 dilution)                                                                ______________________________________                                         .sup.a Legends are same as Table XIV, performed on day 14 postinfection.      .sup.b Vascularization scored as follows: 0 = vessels normal; 1 = some        vessels dilated and infiltrated by minor vessels; 2 = diffuse red with        individual vessels not easily discernable; 3 = diffuse beefy red, vascula     leakage and effusion of blood into conjunctivae.                              .sup.c Discharge scored as follows: 0 = no discharge; 1 = discharge with      moistening of lids and hairs adjacent to lids; 2 = discharge with             moistening of lids, hairs and areas adjacent to eyes.                    

                  TABLE XX                                                        ______________________________________                                        TREATMENT OF LETHAL LCM VIRUS                                                 INFECTION IN MICE.sup.a                                                                            Virus Recovered from Spleen                              Group      Lethality.sup.c                                                                         (PFU × 10.sup.5 /ml).sup.c                         ______________________________________                                        Control    5/5       7.0                                                      SPLV-group 5/5       6.9                                                      R-group    5/5       5.2                                                      SPLV-R-Group                                                                             3/5       3.4                                                      ______________________________________                                         .sup.a Two month old mice were each inoculated intracerebrally with a         lethal dose, i.e., 100 PFU of LCM virus in 0.05 ml inocula.                   .sup.b Lethality is expressed as number dead/number in group.                 .sup.c On the fifth day postinfection 2 mice from each group were             sacrificed and their spleens homogenized at a concentration of 1 gm           spleen/20 ml homogenate.                                                 

SPLVs were prepared using 100 mg lipids and 0.3 ml of 100 mg drug/ml inPBS buffer; the entrapment of drug was 10%; (2) the-"R-group" wastreated with a solution of Ribavarin 3 mg/ml in PBS; (3) the"SPLV-group" was treated with buffer-filled SPLVs (i.e., SPLVs preparedas above but without Ribavarin); and (4) the "control group" was treatedwith PBS. On day 5 post-infection 2 mice from each group were sacrificedand their spleens homogenized (2 spleens/group were homogenized in PBSat 1/20 weight per volume buffer). The plague forming units (PFU) per mlwere determined for each suspension. The remaining 5 mice in each groupswere observed for lethality two times daily for 30 days. The results arepresented in Table XX.

Table XX clearly indicates a decrease in lethality and a decrease in thevirus recoverable from the infected animals. We have not yet determinedwhether these results are due to the anti-viral activity of theribavarin which is released from the SPLVs or whether it is due to animmunomodulation of the mouse host during the sustained release ofribavarin from the SPLVs.

What is claimed is:
 1. A method for preparing stable plurilamellarvesicles, comprising:(a) forming a dispersion of at least oneamphipathic lipid in an organic solvent; (b) combining the dispersionwith a sufficient amount of an aqueous phase to form a biphasic mixturein which the aqueous phase can be completely emulsified; and (c)concurrently emulsifying the aqueous phase while evaporating the organicsolvent of the biphasic mixtures,wherein the stable plurilamellarvesicles produced are substantially free of MLVs, SUVs, and REVs.
 2. Themethod according to claim 1, wherein the ratio of volume of solvent tovolume of aqueous phase is from about 3:1 to about 100:1.
 3. The methodaccording to claim 1, wherein the temperature at which the method isperformed is from about 4° C. to about 60° C.
 4. The method according toclaim 1, wherein the temperature at which the method is performed isless than the phase transition temperature of at least one of saidlipids.
 5. The method according to claim 1, wherein the solvent isfluorocarbon or diethylether, or mixtures thereof.
 6. The methodaccording to claim 5 wherein the solvent contains an anti-oxidant. 7.The method according to claim 6, wherein said anti-oxidant is butylatedhydroxytoluene.
 8. The method according to claim 1 wherein a material tobe entrapped in the vesicles is added with the aqueous phase.
 9. Themethod according to claim 8, wherein at least 20 percent of saidmaterial is entrapped in the vesicles.
 10. The method according to claim8, wherein said material is a protein.
 11. Stable plurilamellar vesiclescomprising lipid vesicles ranging from about lOO nm to about 10,000 nmin size, characterized by a few to over 100 lipid bilayers enclosingaqueous compartments containing at least one entrapped solute in whichthe lipid bilayers have an ordered molecular architecture creating asupramolecular structure which differs from that of other multilamellarvesicles so that when compared to other multilamellar vesicles composedof the identical lipid and aqueous ingredients, stable plurilamellarvesicles have the following properties:(a) a higher percent entrapmentof solute; (b) a lower buoyant density; (c) a volume about one-thirdlarger; (d) greater stability to auto-oxidation during storage inbuffer; (e) greater stability in body fluids; (f) a larger percentleakage of entrapped solute when exposed to urea, guanidine, or ammoniumacetate; (g) a smaller percent leakage of entrapped solute when exposedto hydrochloric acid or serum; and (h) distribution of entrappedcontents throughout the cytosol of cells when administered to the cellsin culture.
 12. Stable plurilamellar vesicles according to claim 11substantially free of MLVs, SUVs, and REVs.
 13. The stable plurilumellarvesicles of claim 11 in which the lipid bilayers comprise aphospholipid.
 14. The stable plurilamellar vesicles of claim 13 in whichthe phospholipid is amphipathic.
 15. Stable plurilamellar vesiclesaccording to claim 11, wherein the major lipid component of the vesiclesis a phosphatidylcholine.
 16. Stable plurilamellar vesicles according toclaim 11, wherein an anti-oxidant is a component of the vesicle. 17.Stable plurilamellar vesicles according to claim 16 wherein saidanti-oxidant is butylated hydroxytoluene.
 18. Stable plurilamellarvesicles according to claim 12, wherein a protein is entrapped withinthe vesicle.
 19. Stable plurilamellar vesicles according to claim 12,wherein a compound selected from the group consisting of: antibacterialcompounds, antifungal compounds, antiparasitic compounds, and antiviralcompounds is entrapped within the vesicle.
 20. Stable plurilamellarvesicles according to claim 11, wherein a compound selected from thegroup consisting of: tumoricidal compounds, toxins, cell receptorbinding molecules, and immunoglobulins is entrapped within the vesicle.21. Stable plurilamellar vesicles according to claim 11, wherein acompound selected from the group consisting of: anti-inflammatorycompounds, anti-glaucoma compounds, mydriatic compounds, and localanesthetics is entrapped within the vesicle.
 22. Stable plurilamellarvesicles according to claim 11, wherein a compound selected from thegroup consisting of: enzymes, hormones, neurotransmitters,immunomodulators, nucleotides, and cyclic adenosine monophosphate isentrapped within the vesicle.
 23. Stable plurilamellar vesiclesaccording to claim 11, wherein a compound selected from the groupconsisting of: dyes, fluorescent compounds, radioactive compounds, andradio-opaque compounds is entrapped within the vesicle.
 24. A method fordelivery of a compound to cells in vivo, comprising: administering to anorganism stable plurilamellar vesicles of claim 11 containing saidcompound entrapped therein.
 25. The method according to claim 24,wherein said stable plurilamellar vesicles are administered topically,intraperitoneally, intravenously, intramuscularly, subcutaneously orintraauricularly.
 26. A method for treatment of infections in animals orplants, comprising: administering stable plurilamellar vesicles of claim11 containing a compound effective for treating said infection.
 27. Themethod according to claim 26, wherein said infection is intracellular.28. The method according to claim 27 wherein said infection is caused bya parasite.
 29. The method according to claim 28, wherein said infectionis caused by Brucella spp.
 30. The method according to claim 29, whereinsaid administration is intraperitoneal.
 31. The method according toclaim 26, wherein said infection is extracellular.
 32. The methodaccording to claim 31 wherein said infection is caused by bacteria. 33.The method according to claim 32, wherein said infection is caused byStaphylococcus aureus.
 34. The method according to claim 33, whereinsaid administration is intraperitoneal.
 35. The method according toclaim 26, wherein said infection is an ocular infection.
 36. The methodaccording to claim 35, wherein said infection is caused by a Moraxellaspp.
 37. The method according to claim 36, wherein said administrationis topical.
 38. The method according to claim 26, wherein said infectionis caused by a virus.
 39. The method according to claim 38, wherein saidinfection is caused by lymphocytic choriomeningitis virus.
 40. Themethod according to claim 39, wherein said administration isintraperitoneal.
 41. A method for treatment of afflictions in animals orplants requiring sustained release of a compound effective for treatingsaid affliction, comprising: administering stable plurilamellar vesiclesof claim 11 containing said compound.
 42. The method according to claim41, wherein said affliction is an ocular affliction.
 43. A methodaccording to claim 42, wherein said affliction is glaucoma.
 44. Themethod according to claim 43, wherein said administration is topical.